Modified recombinant lysosomal alpha-galactosidase A and aspartylglucoaminidase having low mannose-6-phosphate and high sialic acid

ABSTRACT

The present invention relates to lysosomal enzymes modified by use of cell based methods, a compositions comprising a modified lysosomal enzyme, as well as methods for producing a modified lysosomal enzyme and therapeutic use of such modified lysosomal enzyme. In particular, the present disclosure relates to a modified lysosomal enzyme which has low Man6P and low exposed Mannose and high sialic acid content of alpha2,3 type enabling long circulation time and improved uptake into difficult-to-reach organs like heart, kidney and brain.

CROSS-REFERENCE

This application is a U.S. national phase application of InternationalPCT Patent Application No. PCT/US2019/048854, filed Aug. 29, 2019, whichclaims priority to U.S. Provisional Application No. 62/724,543, filedAug. 29, 2018, which is incorporated by reference herein in itsentirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is GLYD_002_01US_ST25.txt. The text file is 60, wascreated on Feb. 26, 2021, and is being submitted electronically viaEFS-Web.

The present disclosure relates to modified lysosomal enzymes,compositions comprising a modified lysosomal enzyme and cell basedmethods for producing a modified lysosomal enzyme. Furthermore, use of amodified lysosomal enzyme in therapy such as in treatment of a lysosomalstorage disease is disclosed.

Also disclosed are cell based glyco-engineering method for modifyingserum half-life (pharmacokinetics) and tissue distribution of alysosomal alpha-galactosidase enzyme (GLA), wherein the enzyme has lowM6P and high sialic acid capping of alpha2,3 type.

Also disclosed are methods for the treatment of lysosomal storagedisease in mammals wherein the mammal is administered a therapeuticallyeffective amount of isolated, glyco-optimized recombinant lysosomalenzyme whereby said storage disease is relieved in difficult-to-reachorgans

BACKGROUND OF THE INVENTION

Lysosomal Storage Diseases (LSDs)

The lysosomal compartment functions as a catabolic machinery thatdegrades waste material in cells. The degradation involves a number ofhydrolases and transporters specifically localized to the lysosome.

Lysosomal storage diseases are inherited metabolic diseases mainlycaused by lack of a specific lysosomal hydrolase activity resulting inbuild-up of substrate in the cells and eventually pathological symptomsin one or more organs. There are around 50 identified lysosomal storagediseases including, e.g., Gaucher and Fabry diseases, where a link hasbeen established between disease and mutations in genes coding forlysosomal proteins. How the accumulated storage material cause pathologyis not fully understood and varies from one disease to the other.

Enzyme Replacement Therapy (ERT)

Excess storage can be reduced by administration of a lysosomal enzymefrom a heterologous source. ERT's addressing all severe symptoms ofGaucher, Fabry disease or any other LSD are not available. The majorlimitations of ERT is lack of penetrance of the enzyme to keypathological sites or difficult-to-reach organs (for example kidney,heart, brain), leaving a large segment of LSD patients without anytreatment options for their most severe symptoms.

It is well established that intravenous administration of a lysosomalenzyme results in its rapid uptake by cells via a mechanism calledreceptor mediated endocytosis. This endocytosis is mediated by receptorson the cell surface, and in particular the two mannose-6 phosphatereceptors (M6PRs) and the mannose receptor (MR) have been shown to bepivotal for uptake of most lysosomal enzymes (Grubb 2010). The M6PRrecognize phosphorylated oligomannose glycans which are characteristicfor lysosomal proteins. Based on the principle of receptor mediatedendocytosis, enzyme replacement therapies (ERT) are today available fora number of LSDs, including Gaucher type I, Fabry, Pompe, Wolman,Neuronal Ceroid Lipofuscinosis, and the Mucopolysaccharidosis type I,II, IVA, VI and VII diseases. These therapies are efficacious inreducing lysosomal storage in various peripheral organs and therebyameliorate some symptoms related to the pathology. A majority of theLSDs however cause lysosomal storage in organs that are difficult toreach via the receptor mediated endocytic route, such difficult-to-reachorgans include central nervous system (CNS), heart, kidney and muscle,and consequently many patients present a repertoire of serious signs andsymptoms that cannot be adequately addressed with current treatments. Amajor drawback with intravenously administered ERT's is the poordistribution to the difficult-to-reach organs. The CNS for example isprotected from exposure to blood borne compounds by theblood-brain-barrier, formed by the CNS endothelium, and since 70% ofLSD's present as progressive neurodegenerative diseases (Platt 2018)many LSD patients will eventually face mental deterioration, oftenassociated with poor/short life expectancy. For Fabry disease thecurrent GLA enzyme treatment options does not effectively alleviatesymptoms in kidney and heart and patients inevitably presentsprogressively severe symptoms in those organs (Desnick 2012).

Thus, the lysosomal replacement enzyme treatments available in the arthave therapeutic deficiencies that differ among diseases, but in generalthere is insufficient or no enzyme delivery to all necessary sites ofpathology; inability of the therapeutic enzyme to reach certainsanctuary sites in periphery nor pass blood-brain barrier, which causesincreasingly severe CNS symptoms for most of the lysosomal diseases(Kishnani 2015, Platt 2018). Moreover, many current lysosomalreplacement enzymes have very short circulation half-life. Thus there isneed for ERT's with improved biodistribution and systems for screeningand identifying such improved enzymes and for bringing such enzymes tobenefit of patients cost-effective mammalian production systems areneeded.

The present invention provides recombinant lysosomal enzymes, producedin glycoengineered mammalian cells, with modified glycostructures. Therecombinant lysosomal enzymes comprise one or more glycans that havebeen modified compared to the glycans of the lysosomal enzyme naturallyproduced in humans or in standard mammalian cells and the resultantenzymes have various improved properties including, e.g., improvedcirculation time and/or improved targeting to hard-to-reach organs, suchas kidney, heart, muscle or brain.

Glycosylation of lysosomal enzymes

In general, N-glycosylations can occur at a Asn-X-Ser/Thr sequencemotif. To this motif the initial dolichol-linked oligosaccharideprecursor is transferred by the glycosyltransferaseoligosaccharyltransferase, within the lumen of the ER. This common basisfor all N-linked glycans is made up of 14 residues; 3 glucose, 9mannose, and 2 N-acetylglucosamine (FIG. 1 ). This ancestor is thenconverted into three general types of N-glycans: oligomannose, complexand hybrid, by the actions of a multitude of enzymes that both trim downthe initial precursor and add new sugar moieties. Each mature N-glycancontains the common core Man(Man)2-GlcNAc-GlcNAc-Asn, where Asn is theattachment point to the protein. In addition, proteins directed to thelysosome carry one or more N-glycans which are phosphorylated. Thephosphorylation occurs in the Golgi and is initiated by the addition ofN-acetylglucosamine-1-phosphate to C-6 of mannose residues ofoligomannose type N-glycans, a process catalyzed by thephospho-transferase complex comprising GNPTG and GNPTAB proteins (FIG. 1). The N-Acetylglucosamine is cleaved off to generate exposedMannose-6-phospate (Man6P) residues, which are recognized by M6PRs andwill facilitate the transport of the lysosomal protein to the lysosome.The resulting N-glycan is then trimmed to the point where the Man6P isthe terminal group of the N-glycan chain. (Essentials of Glycobiology.3^(rd) edition. Varki A, Cummings R D, Esko J D, et al, editors. ColdSpring Harbor (N. Y.): Cold Spring Harbor Laboratory Press; 2017). Thebinding site of the M6PR requires a terminal Man6P group that iscomplete, as both the sugar moiety and the phosphate group is involvedin the binding to the receptor (Kim 2009). The binding of Man6P taggedenzyme to cell based M6PRs has been considered critical for cellularuptake and subsequent pharmaceutical effect of nearly all ERT's fromcirculation after exogenous administration via iv route (Grubb 2010).Accordingly, typical strategies for producing ERT focus on ensuring highmannose or mannose-6 phosphate content.

The need for glycosylation on ERT's makes mammalian cells the preferredproduction platform, in particular the Chinese hamster ovary (CHO) cellsare used extensively for production. However, use of yeast and plantcell systems have been utilized to enhance Man6P and/or exposed Mancontent of lysosomal enzymes in order to increase uptake by MR and M6PR.Mammalian cells such as CHO cells produce heterogeneous glycosylation oflysosomal enzymes contained variable degrees of Man6P and/or exposed Mancontent and complex N-glycans with sialic acids.

SUMMARY OF THE INVENTION

In some embodiments, the present disclosure relates to lysosomal enzymesproduced in glycoengineered mammalian cells, wherein one or more glycanshave been modified compared to glycans of the lysosomal enzyme producedin man or standard mammalian cells.

An object of the present invention is to use engineered mammalian celllines with different glycosylation capacities to produce lysosomalenzymes with glycostructures that prolong serum half-life and/or changetissue targeting of the enzyme in mammals and thus improve efficacy ofthe enzyme for treating human disease.

An object of the present invention relates to use of mammalian cellswith modified glycosylation capacities for producing lysosomal enzymeswith improved biodistribution and efficacy for treating human disease.

An object of the present invention relates to methods to producelysosomal enzymes without M6P and/or without high-Man content and withsialic acid capped N-glycans.

An object of the present invention relates to methods to producelysosomal enzymes without M6P and/or without high-Man content and withalphα2-3 linked sialic acid capped N-glycans.

An object of the present invention relates to use of lysosomal enzymeswithout M6P and/or without high-Man content and with sialic acid cappedN-glycans.

An object of the present invention relates to use of lysosomal enzymeswithout M6P and/or without high-Man content and with alphα2-3 linkedsialic acid capped N-glycans.

In one embodiment, the present disclosure provides a modifiedrecombinant lysosomal enzyme with increased circulation time in plasmaas compared to an unmodified version of the same, said enzyme beingproduced in a mammalian cell line with modified glycosylation capacitydue to altered expression of one or more genes involved inglycosylation. In some embodiments, the modified recombinant lysosomalenzyme comprises less than 10% mannose-6-phosphate (Man6P) and less than0.3 mole exposed mannose (Man) per mole of enzyme. In some embodiments,the modified recombinant lysosomal enzyme comprises more than 4 molsialic acid (SA) per mol of enzyme. In some embodiments, the modifiedrecombinant lysosomal enzyme comprises more than 4 mol alpha2,3SA permol of enzyme. In some embodiments, the modified recombinant lysosomalenzyme comprises more than 4.5 mol alpha2,3SA per mol of enzyme. In someembodiments, the modified recombinant lysosomal enzyme comprises morethan 4 mol alpha2,3SA per mol of enzyme and less than 1 mol alpha2,6SA.In some embodiments, the modified recombinant lysosomal enzyme compriseshomogeneous N glycans:

-   -   a) with alpha2,3SA capping,    -   b) without alpha2,3SA capping,    -   c) with alpha2,6SA capping,    -   d) without alpha2,6SA capping,    -   e) with <0.3 mol Man6P per mole of enzyme, and    -   h) with <0.1 mol Man6P per mole of enzyme.        In some embodiments, the mammalian cells are engineered to        produce modified recombinant lysosomal enzyme with high 2,3        sialic acid capping. In some embodiments, the mammalian cells        are engineered to produce modified recombinant lysosomal enzyme        with biantennary N-glycan structures, tri-antennary N-glycan        structures, and/or tetra-antennary, N-glycan structures. In some        embodiments, the mammalian cells are engineered to produce        modified recombinant lysosomal enzyme containing greater than        90% homogeneity for biantennary N-glycan structures. In some        embodiments, the mammalian cells are engineered to produce        modified recombinant lysosomal enzyme containing greater than        90% homogeneity for tri or tetra-antennary N-glycan structures.        In some embodiments, the mammalian cells are engineered to        produce modified recombinant lysosomal enzyme containing greater        than 90% homogeneity for tetra-antennary N-glycan structures. In        some embodiments, the modified recombinant lysosomal enzyme is        selected from Aspartylglucoaminidase (AGA), Alpha-Galactosidase        A (GLA), Acid ceramidase, Acid alpha-L-fucosidase, Protective        protein/Cathepsin A, Acid beta-glucosidase, or        glucocerebrosidase (GBA), Acid beta-galactosidase,        Iduronate-2-sulfatase (IDS), Alpha-L-Iduronidase (IDUA),        Galactocerebrosidase/galactosylceramidase (GALC), Acid        alpha-mannosidase, Acid beta-mannosidase, Arylsulfatase B,        Arylsulfatase A, Acid beta-galactosidase,        N-Acetylglucosamine-1-phosphotransferase, and Lysosomal        alpha-glucosidase (GAA). In some embodiments, the modified        recombinant lysosomal enzyme is selected from GLA, GBA, GUS, and        GAA. In some embodiments, the modified recombinant lysosomal        enzyme is GLA. In some embodiments, the modified recombinant        lysosomal enzyme is GBA. In some embodiments, the mammalian        cells are engineered to produce modified recombinant lysosomal        enzyme with no M6P, high 2,3SA, and, optionally, a biantennary,        triantennary, or tetra antennary N glycan structure. In some        embodiments, the modified recombinant lysosomal enzyme comprises        lowered mannose-6-phosphate (M6P) tagging of N-glycans as        compared to a similar unmodified recombinant lysosomal enzyme.        In some embodiments, the modified recombinant lysosomal enzyme        comprises increased glycosylation homogeneity as compared to a        similar unmodified recombinant lysosomal enzyme. In some        embodiments, the modified recombinant lysosomal enzyme comprises        any glycosylation pattern that is without fucose. In some        embodiments, the glycoengineered mammalian host cell comprises        one or more inactivation of an endogenous glycogene and/or one        or more introduction of an exogenous glycogene, wherein said        glycogenes are selected from the group consisting of GNPTAB,        GNPTG, NAGPA, ALG3, ALG5, ALG6, ALG8, ALG9, ALG10, ALG12,        Mannosidases, MAN1A1, MAN1A2, MAN1B1, MAN1C1, MAN2A1, MAN2A2,        MOGS, GANAB, MGAT1, MGAT2 and Sialyl transferases. In some        embodiments, the glycoengineered mammalian host cell comprises        one or more inactivation of an endogenous glycogene and/or one        or more introduction of an exogenous glycogene, wherein the        endogenous glycogene is one or more of St3gal4 and St3gal6. In        some embodiments, the glycoengineered mammalian host cell        comprises a knockout of GNPTAB and/or GNPTG. In some        embodiments, the glycoengineered mammalian host cell comprises a        knock-in of St3gal4 and/or St3gal6. In some embodiments, the        glycoengineered mammalian host cell comprises a knockout of        Mgat4b and/or Mgat5. In some embodiments, the glycoengineered        mammalian host cell comprises a knockout of GNPTG and/or GNPTAB        and a knock-in of St3gal4 and/or St3gal6. In some embodiments,        the mammalian cells comprise a knockout of one or more of GNPTG,        GNPTAB, Mgat4b, Mgat5 and a knock-in of one or more of St3gal4        and St3gal6. In some embodiments, the mammalian cells comprise a        knockout of GNPTG, GNPTAB, Mgat4b, Mgat5 and a knock-in of        St3gal4. In some embodiments, the cell is selected from the        group consisting of CHO, HEK293, NS0, SP2/0, YB2/0, HUVEC, HKB,        PER-C6, NS0, or derivatives of any of these cells. In some        embodiments, the cell is a CHO cell or derivative thereof,        wherein optionally the cell is a CHO cell derivative selected        from CHO-K1, CHO DXB11, CHO-S, CHO-DG44 and CHO-GS.

In one embodiment, the present disclosure provides a modifiedrecombinant lysosomal enzyme produced by a glycoengineered mammalianhost cell, said enzyme comprising increased circulation time in plasmaas compared to an unmodified version of the same enzyme, wherein saidincreased circulation time is due to the carbohydrate moieties added tothe modified recombinant lysosomal enzyme by one or moreglycosyltransferase enzyme expressed in the glycoengineered mammalianhost, wherein the carbohydrate moieties reduce binding of the enzyme tomannose receptors and/or mannose 6-phosphate receptors, and wherein themodified recombinant lysosomal enzyme retains enzymatic activity. Insome embodiments, the mammalian cells are engineered to produce modifiedrecombinant lysosomal enzyme with high 2,3 sialic acid capping. In someembodiments, the mammalian cells are engineered to produce modifiedrecombinant lysosomal enzyme with biantennary N-glycan structures,tri-antennary N-glycan structures, and/or tetra-antennary, N-glycanstructures. In some embodiments, the mammalian cells are engineered toproduce modified recombinant lysosomal enzyme containing greater than90% homogeneity for biantennary N-glycan structures. In someembodiments, the mammalian cells are engineered to produce modifiedrecombinant lysosomal enzyme containing greater than 90% homogeneity fortri or tetra-antennary N-glycan structures. In some embodiments, themammalian cells are engineered to produce modified recombinant lysosomalenzyme containing greater than 90% homogeneity for tetra-antennaryN-glycan structures. In some embodiments, the modified recombinantlysosomal enzyme is selected from Aspartylglucoaminidase (AGA),Alpha-Galactosidase A (GLA), Acid ceramidase, Acid alpha-L-fucosidase,Protective protein/Cathepsin A, Acid beta-glucosidase, orglucocerebrosidase (GBA), Acid beta-galactosidase, Iduronate-2-sulfatase(IDS), Alpha-L-Iduronidase (IDUA),Galactocerebrosidase/galactosylceramidase (GALC), Acidalpha-mannosidase, Acid beta-mannosidase, Arylsulfatase B, ArylsulfataseA, Acid beta-galactosidase, N-Acetylglucosamine-1-phosphotransferase,and Lysosomal alpha-glucosidase (GAA). In some embodiments, the modifiedrecombinant lysosomal enzyme is selected from GLA, GBA, GUS, and GAA. Insome embodiments, the modified recombinant lysosomal enzyme is GLA. Insome embodiments, the modified recombinant lysosomal enzyme is GBA. Insome embodiments, the mammalian cells are engineered to produce modifiedrecombinant lysosomal enzyme with no M6P, high 2,3SA, and, optionally, abiantennary, triantennary, or tetra antennary N glycan structure. Insome embodiments, the modified recombinant lysosomal enzyme compriseslowered mannose-6-phosphate (M6P) tagging of N-glycans as compared to asimilar unmodified recombinant lysosomal enzyme. In some embodiments,the modified recombinant lysosomal enzyme comprises increasedglycosylation homogeneity as compared to a similar unmodifiedrecombinant lysosomal enzyme. In some embodiments, the modifiedrecombinant lysosomal enzyme comprises any glycosylation pattern that iswithout fucose. In some embodiments, the glycoengineered mammalian hostcell comprises one or more inactivation of an endogenous glycogeneand/or one or more introduction of an exogenous glycogene, wherein saidglycogenes are selected from the group consisting of GNPTAB, GNPTG,NAGPA, ALG3, ALG5, ALG6, ALG8, ALG9, ALG10, ALG12, Mannosidases, MAN1A1,MAN1A2, MAN1B1, MAN1C1, MAN2A1, MAN2A2, MOGS, GANAB, MGAT1, MGAT2 andSialyl transferases. In some embodiments, the glycoengineered mammalianhost cell comprises one or more inactivation of an endogenous glycogeneand/or one or more introduction of an exogenous glycogene, wherein theendogenous glycogene is one or more of St3gal4 and St3gal6. In someembodiments, the glycoengineered mammalian host cell comprises aknockout of GNPTAB and/or GNPTG. In some embodiments, theglycoengineered mammalian host cell comprises a knock-in of St3gal4and/or St3gal6. In some embodiments, the glycoengineered mammalian hostcell comprises a knockout of Mgat4b and/or Mgat5. In some embodiments,the glycoengineered mammalian host cell comprises a knockout of GNPTGand/or GNPTAB and a knock-in of St3gal4 and/or St3gal6. In someembodiments, the mammalian cells comprise a knockout of one or more ofGNPTG, GNPTAB, Mgat4b, Mgat5 and a knock-in of one or more of St3gal4and St3gal6. In some embodiments, the mammalian cells comprise aknockout of GNPTG, GNPTAB, Mgat4b, Mgat5 and a knock-in of St3gal4. Insome embodiments, the cell is selected from the group consisting of CHO,HEK293, NS0, SP2/0, YB2/0, HUVEC, HKB, PER-C6, NS0, or derivatives ofany of these cells. In some embodiments, the cell is a CHO cell orderivative thereof, wherein optionally the cell is a CHO cell derivativeselected from CHO-K1, CHO DXB11, CHO-S, CHO-DG44 and CHO-GS.

In one embodiment, the present disclosure provides an engineeredmammalian cell line, comprising a knockout of one or both of Gnptg andgnptab. In some embodiments, the engineered mammalian cell linecomprises a knock-in of one or both of St3gal4 and St3gal6. In someembodiments, the engineered mammalian cell line comprises a knockout ofone or both of Mgat4b and Mgat5. In some embodiments, the engineeredmammalian cell line comprises a knockout of one or both of Mgat4b andMgat5 and a knock-in of one or both of St3gal4 and St3gal6. In someembodiments, the engineered mammalian cell line comprises a knock-in ofone or more of St3gal4, St3gal6, Mgat4a, Mgat4b, and Mgat5. In someembodiments, the engineered mammalian cell line comprises a knockout ofGNPTG, GNPTAB, Mgat4b, Mgat5 and a knock-in of St3gal4. In someembodiments, the engineered mammalian cell line is selected from thegroup consisting of CHO, HEK293, NS0, SP2/0, YB2/0, HUVEC, HKB, PER-C6,NS0, or derivatives of any of these cells. In some embodiments, theengineered mammalian cell line is CHO cell or derivative thereof. Insome embodiments, the cell line has been engineered to produce glycanswith one or more of the following:

-   -   a) with alpha2,3SA capping,    -   b) without alpha2,3SA capping,    -   c) with alpha2,6SA capping,    -   d) without alpha2,6SA capping,    -   e) low Man6P (<0.3 mol per mol of enzyme), and    -   h) no Man6P (<0.05 mol per mol of enzyme)        In some embodiments, the cell produces a modified lysosomal        enzyme. In some embodiments, the cell produces any one of the        modified recombinant lysosomal enzymes disclosed herein.

In one embodiment, the present disclosure provides a mammalian cell linethat comprises one or more endogenous glycogene inactivated and/orexogenous glycogene introduced said glycogenes selected from the list ofGNPTAB, GNPTG, NAGPA, ALG3, ALG5, ALG6, ALG8, ALG9, ALG10, ALG12,Mannosidases, MAN1A1, MAN1A2, MAN1B1, MAN1C1, MAN2A1, MAN2A2, MOGS,GANAB, MGAT1, MGAT2 and Sialyl transferases. In one embodiment, thepresent disclosure provides a mammalian cell line producing any one ormore of the modified recombinant lysosomal enzymes disclosed herein,wherein said cell line has one or more endogenous glycogene inactivatedand/or exogenous glycogene introduced said glycogenes selected from thelist of GNPTAB, GNPTG, NAGPA, ALG3, ALG5, ALG6, ALG8, ALG9, ALG10,ALG12, Mannosidases, MAN1A1, MAN1A2, MAN1B1, MAN1C1, MAN2A1, MAN2A2,MOGS, GANAB, MGAT1, MGAT2 and Sialyl transferases. In some embodiments,the sialyl transferase comprises one or more of St3gal4 and St3gal6. Insome embodiments, the cell comprises an inactivated GNPTAB gene. In someembodiments, the modified recombinant lysosomal enzyme containsincreased sialic acids due to the inactivation of said inactivatedGNPTAB gene. In some embodiments, one or more cells of the cell linecontains an introduction of one or more glycosyltransferase geneselected from the group consisting of MGAT4A, MGAT4B, MGAT5 and MGAT5B.In some embodiments, one or more cells of said cell line contains aninactivation and/or introduction of one or more glycogene selected fromthe group consisting of ALG3, ALG6, ALG8, ALG9, ALG10, and ALG12. Insome embodiments, one or more cells of said cell line contains aninactivation and/or introduction of one or more glycogene selected fromthe group consisting of MGAT2, MGAT4A, MGAT4B, MGAT5, and MGAT5B. Insome embodiments, one or more cells of said cell line contains aninactivation and/or introduction of one or more glycogene selected fromthe group consisting of FUT8, ST3GAL4/6, ST6GAL1/2. In some embodiments,the mammalian cell is selected from the group consisting of CHO, HEK293,In some embodiments, the cell is a CHO cell or derivative thereof.

In some embodiments, the present disclosure provides a method forproducing a lysosomal enzyme in a cell, wherein the lysosomal enzymecomprises a modified glycan profile, and wherein the cell producing thelysosomal enzyme has more than one modification of one or moreglycogenes. In one embodiment, the cells have been modified by glycogeneknock-out and/or knock-in of an exogenous DNA sequence coding for aglycosyltransferase.

In some embodiments, the present disclosure provides a mammalian cellcapable of expressing a lysosomal enzyme, wherein the enzyme comprisesone or more of the posttranslational modification patterns:

-   -   a) with alpha2,3SA capping,    -   b) without alpha2,3SA capping,    -   c) with alpha2,6SA capping,    -   d) without alpha2,6SA capping,    -   e) with low Man6P (<0.3 mol Man6P per mole of enzyme), or    -   h) with no Man6P (<0.05 mol Man6P per mole of enzyme)

In some embodiments, the present disclosure provides a method ofproducing a modified recombinant enzyme disclosed herein in a cell lineaccording to the present disclosure, wherein the cell line furthercomprises an expression vector encoding the coding sequence of therecombinant enzyme. In some embodiments, the enzyme is selected fromAspartylglucoaminidase (AGA), Alpha-Galactosidase A (GLA), Acidceramidase, Acid alpha-L-fucosidase, Protective protein/Cathepsin A,Acid beta-glucosidase, or glucocerebrosidase (GBA), Acidbeta-galactosidase, Iduronate-2-sulfatase (IDS), Alpha-L-Iduronidase(IDUA), Galactocerebrosidase/galactosylceramidase (GALC), Acidalpha-mannosidase, Acid beta-mannosidase, Arylsulfatase B, ArylsulfataseA, Acid beta-galactosidase, N-Acetylglucosamine-1-phosphotransferase,and Lysosomal alpha-glucosidase (GAA). In some embodiments, the modifiedrecombinant lysosomal enzyme is selected from GLA, GBA, GUS, and GAA Insome embodiments, the modified recombinant lysosomal enzyme is GLA. Insome embodiments, the modified recombinant lysosomal enzyme is GBA.

In some embodiments, the present disclosure provides a method oftreating a lysosomal storage disorder with the modified recombinantlysosomal enzyme disclosed herein. In some embodiments, the presentdisclosure provides any one or more of the modified recombinantlysosomal enzyme disclosed herein for use in a therapy. In someembodiments, the enzyme is fused to a non-glycan tag designed to improveblood-brain-barrier passage. In some embodiments, the treatment is of amammal afflicted with a lysosomal storage disease.

In some embodiments, the present disclosure provides a compositioncomprising a substantially pure preparation of a modified recombinantlysosomal enzyme disclosed herein. In some embodiments, the compositionfurther comprises one or more pharmaceutically acceptable carrier,excipient, diluent, and/or surfactant. In some embodiments, thecomposition further comprises a compound that improves the serumstability of the enzyme. In some embodiments, the composition furthercomprises DGJ.

In some embodiments, the present disclosure provides a method oftreating a mammal afflicted with a lysosomal storage disease, comprisingadministering to the mammal a therapeutically effective amount of amodified lysosomal enzyme, said modified enzyme being selected from:

-   -   a) a modified recombinant lysosomal enzyme with increased        circulation time in plasma as compared to an unmodified version        of the same, said enzyme being produced in a mammalian cell line        with modified glvcosvlation capacity due to altered expression        of one or more genes involved in glvcosvlation;    -   b) a lysosomal enzyme composition that is a substantially pure        preparation of the modified recombinant lysosomal enzyme of (a),    -   and    -   c) a modified lysosomal enzyme wherein the modification        comprises reduction of M6P content and increase of NeuAc capping        of the glycans, whereby the enzyme has reduced its activity with        respect to glycan recognition receptors and increased        circulatory half-life and better targeting to one or more of the        difficult-to-reach organs like brain, kidney, heart and muscle.        In some embodiments, the modified recombinant lysosomal enzyme        is selected from Aspartylglucoaminidase (AGA),        Alpha-Galactosidase A (GLA), Acid ceramidase, Acid        alpha-L-fucosidase, Protective protein/Cathepsin A, Acid        beta-glucosidase, or glucocerebrosidase (GBA), Acid        beta-galactosidase, Iduronate-2-sulfatase (IDS),        Alpha-L-Iduronidase (IDUA),        Galactocerebrosidase/galactosylceramidase (GALC), Acid        alpha-mannosidase, Acid beta-mannosidase, Arylsulfatase B,        Arylsulfatase A, Acid beta-galactosidase,        N-Acetylglucosamine-1-phosphotransferase, and Lysosomal        alpha-glucosidase (GAA). In some embodiments, the modified        recombinant lysosomal enzyme is selected from GLA, GBA, GUS, and        GAA. In some embodiments, the modified recombinant lysosomal        enzyme is GLA. In some embodiments, the modified recombinant        lysosomal enzyme is GBA or GUS. In some embodiments, the        lysosomal storage disease is selected from        Aspartylglucoaminouria (AGU), Fabry, Farber, Fucosidosis,        Galactosidosialidosis, Gaucher types 1,2, and 3, G-M1        gangliosidosis, Hunter, Hurler-Scheie, Krabbe,        Alpha-Mannosidosis (Laman), Beta-Mannosidosis, Maroteaux-Lamy,        Metachromatic, Morquio B, Mucolipidosis II/III, and Pompe.

In some embodiments, the present disclosure provides a recombinant CHOcell line comprising a knockout of each of the Gnptab, Gnptg, Mgat4b,and Mgat5 genes and further comprising a knock-in of the ST3gal4 gene,for use in the in vitro production of a recombinant lysosomal enzymecontaining homogenous N glycans containing less than 0.3 mol exposedmannose, less than 0.3 mol exposed mannose 6 phosphate, and greater than4 mol terminal alpha2,3, sialylation per mol of enzyme. In someembodiments, the recombinant lysosomal enzyme has less than 0.5 molalpha2,6, sialylation per mol of enzyme. In some embodiments, therecombinant lysosomal enzyme has less than 0.1 mol alpha2,6, sialylationper mol of enzyme. In some embodiments, the e recombinant lysosomalenzyme is selected from Aspartylglucoaminidase (AGA),Alpha-Galactosidase A (GLA), Acid ceramidase, Acid alpha-L-fucosidase,Protective protein/Cathepsin A, Acid beta-glucosidase, orglucocerebrosidase (GBA), Acid beta-galactosidase, Iduronate-2-sulfatase(IDS), Alpha-L-Iduronidase (IDUA),Galactocerebrosidase/galactosylceramidase (GALC), Acidalpha-mannosidase, Acid beta-mannosidase, Arylsulfatase B, ArylsulfataseA, Acid beta-galactosidase, N-Acetylglucosamine-1-phosphotransferase,and Lysosomal alpha-glucosidase (GAA). In some embodiments, therecombinant lysosomal enzyme is GLA.

In some embodiments, the present disclosure provides a method forincreasing the biodistribution of a recombinant lysosomal enzyme to adifficult to reach organ, wherein the lysosomal enzyme comprises one ormore N-glycan, the method comprising glycoengineering the recombinantlysosomal enzyme to homogeneously contain N-glycan structures containinglow levels of exposed mannose and/or low levels of exposedmannose-6-phosphate glycans. In some embodiments, the glycoengineeringis achieved by producing the recombinant lysosomal enzyme in a mammaliancell comprising one or more inactivation of an endogenous glycogeneand/or one or more introduction of an exogenous glycogene. In someembodiments, the method further comprises glycoengineering therecombinant lysosomal enzyme to contain N-glycan structures with highlevels of terminal sialyation. In some embodiments, the terminalsialylation is primarily alpha2,3 type. In some embodiments, theN-glycan structures contain less than 5%, less than 1%, or no detectableN-glycan structures with exposed mannose and/or exposedmannose-6-phosphate glycans. In some embodiments, the N-glycanstructures contain less than 0.5 mol, less than 0.1 mol, or nodetectable alpha2,6 type terminal sialylation per mol of enzyme. In someembodiments, greater than 75% or greater than 90% of the N-glycanstructures are biantennary. In some embodiments, greater than 90% of theN-glycan structures are biantennary due to the inactivation of one ormore branching enzyme normally expressed in the mammalian cell. In someembodiments, greater than 75% or greater than 90% of the N-glycanstructures are tri- or tetra-antennary. In some embodiments, therecombinant lysosomal enzyme exhibits enzyme activity in plasma that isequal to or greater than the enzyme activity of a similar enzyme lackingsaid glycoengineering. In some embodiments, the recombinant lysosomalenzyme exhibits enzyme activity in a difficult to reach organ that isequal to or greater than the enzyme activity of a similar enzyme lackingsaid glycoengineering. In some embodiments, the recombinant lysosomalenzyme exhibits a serum half-life that is equal to or greater than theserum half-life of a similar enzyme lacking said glycoengineering. Insome embodiments, the recombinant lysosomal enzyme exhibits a bloodclearance that is equal to or greater than the blood clearance of asimilar enzyme lacking said glycoengineering. In some embodiments, thedifficult to reach organ is the heart. In some embodiments, thedifficult to reach organ is the kidney. In some embodiments, thedifficult to reach organ is the liver.

In some embodiments, the present disclosure provides an unpurifiedmammalian cell culture supernatant, the cell culture comprising apopulation of mammalian cells engineered to produce recombinantlysosomal enzyme with a glycosylation pattern that is more homogenousthan a glycosylation pattern of the same enzyme when produced inmammalian cells that have not been similarly engineered, wherein themammalian cells comprise at least a knockout of GNPTAB and GNPTG. Insome embodiments, the mammalian cells further comprise a knockout ofMgat4b and/or Mgat5. In some embodiments, the mammalian cells comprise aknock-in of St3gal4. In some embodiments, the mammalian cells comprise aknock-in of St6gal1. In some embodiments, the mammalian cells furthercomprise a knockout of St3gal4 and/or St3gal6. In some embodiments, themammalian cells comprise a knockout of one or more of GNPTG, GNPTAB,Mgat4b, Mgat5 and a knock-in of one or more of St3gal4 and St3gal6. Insome embodiments, the unpurified mammalian cell culture supernatantcomprises recombinant lysosomal enzyme containing substantiallyhomogeneous glycosylation. In some embodiments, the unpurified mammaliancell culture supernatant comprises mammalian cells that are engineeredto produce recombinant lysosomal enzyme: a) with alpha2,3SA capping,

-   -   b) without alpha2,3SA capping,    -   c) with alpha2,6SA capping,    -   d) without alpha2,6SA capping,    -   e) low Man6P, and    -   h) no Man6P (<1%)        In some embodiments, the mammalian cells are engineered to        produce recombinant lysosomal enzyme with less than <0.3 mol        Man6P per mol of enzyme. In some embodiments, the mammalian        cells are engineered to produce recombinant lysosomal enzyme        with less than <0.05 mol Man6P per mol of enzyme. In some        embodiments, the mammalian cells are engineered to produce        recombinant lysosomal enzyme with high 2,3 sialic acid capping.        In some embodiments, the mammalian cells are engineered to        produce recombinant lysosomal enzyme containing biantennary        N-glycan structures, tri-antennary N-glycan structures, and/or        tetra-antennary, N-glycan structures. mammalian cells are        engineered to produce recombinant lysosomal enzyme containing        greater than 90% homogeneity for biantennary N-glycan        structures. In some embodiments, the mammalian cells are        engineered to produce recombinant lysosomal enzyme containing        greater than 90% homogeneity for tri-antennary N-glycan        structures. In some embodiments, the mammalian cells are        engineered to produce recombinant lysosomal enzyme containing        greater than 90% homogeneity for tri-antennary N-glycan        structures. In some embodiments, the mammalian cells are        selected from the group consisting of CHO, HEK293, NS0, SP2/0,        YB2/0, HUVEC, HKB, PER-C6, NS0, or derivatives of any of these        In some embodiments, the modified lysosomal enzyme is selected        from Aspartylglucoaminidase (AGA), Alpha-Galactosidase A (GLA),        Acid ceramidase, Acid alpha-L-fucosidase, Protective        protein/Cathepsin A, Acid beta-glucosidase, or        glucocerebrosidase (GBA), Acid beta-galactosidase,        Iduronate-2-sulfatase (IDS), Alpha-L-Iduronidase (IDUA),        Galactocerebrosidase/galactosylceramidase (GALC), Acid        alpha-mannosidase, Acid beta-mannosidase, Arylsulfatase B,        Arylsulfatase A, Acid beta-galactosidase,        N-Acetylglucosamine-1-phosphotransferase, and Lysosomal        alpha-glucosidase (GAA). In some embodiments, the modified        lysosomal enzyme is selected from GLA, GBA, GUS, and GAA. In        some embodiments, the modified lysosomal enzyme is GLA. In some        embodiments, the modified lysosomal enzyme is GBA. In some        embodiments, the cell culture comprising a population of        mammalian cells engineered to produce recombinant lysosomal        enzyme with a glycosylation pattern with a high mannose        glycosylation content as compared to the same enzyme when        produced in mammalian cells that have not been similarly        engineered, wherein the mammalian cells comprise at least a        knockout of Alg3 or Alg9 and at least a knock-in of GNPTAB. In        some embodiments, the unpurified mammalian cell culture        supernatant further comprises a knock-in of GNPTG.

In some embodiments, the present disclosure provides a compositioncomprising a substantially pure preparation of a lysosomal enzymeisolated from an unpurified mammalian cell culture supernatant disclosedherein or comprising a modified recombinant lysosomal enzyme disclosedherein. In some embodiments, the composition further comprises one ormore pharmaceutically acceptable carrier, excipient, diluent, and/orsurfactant. In some embodiments, the composition further comprises acompound which improves the serum stability of the enzyme. In someembodiments, the composition further comprises DGJ. In some embodiments,the lysosomal enzyme is fused to a non-glycan tag designed to improveblood-brain-barrier passage. In some embodiments, the non-glycan tag isselected from the group consisting of IGF2, Transferrin, Immunoglobinand a fragment or derivative thereof. In some embodiments, thecomposition further comprises α-Gal A and DGJ or a pharmaceuticallyacceptable salt thereof. In some embodiments, the composition isformulated for parenteral administration to a subject. In someembodiments, the composition is formulated for administration in anamount of 0.3, 0.5, 1, 2 or 3 mg/kg. In some embodiments, thecomposition is formulated for administration in an amount of 0.1, 0.3,0.5, 1, 3, or 10 mg/kg. In some embodiments, the composition is for usein the preparation of a medicament for the treatment of Fabry disease.In some embodiments, the composition is for use in the treatment ofFabry disease. In some embodiments, the composition is for use in thepreparation of a medicament for the treatment of a lysosomal storagedisease. In some embodiments, the composition is for use in thetreatment of a lysosomal storage disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows major CHO genes involved in N-glycan processing. The corestructure of 9Man and 2GlcNAc is shown on the left and the Man6Pcontaining glycans and complex type glycans are to the top-right andlower-right respectively. Some genes addressed in the currentapplication are in bold (Table 1).

FIG. 2 shows site specific glyco-analysis of GLA expressed in wt CHOcells and five engineered cell lines. The two most abundant glycanstructures at N-glycosites N108, N161 and N184 of GLA are shown, withFabrazyme included as control.

FIG. 3 shows enzyme activity in plasma at various time points afterinjection of GLA. Fabry mice were injected with 1 mg/kg dose of wt-CHOproduced GLA or Fabrazyme via tail vein (n=4 per group, male mice, age3.5 months). At indicated time points, small amount of blood sampleswere collected from tail vein and separated plasma was analyzed for GLAactivity. Enzyme activities were shown as % of activity at 5 min afterinjection. Data are expressed as mean f S.D. (n=4). The figure show verysimilar plasma clearance curves for Fabrazyme and GLA produced inwt-CHO.

FIG. 4 shows tissue distribution of infused enzymes in Fabry mice. Heart(FIG. 4A), kidney (FIG. 4B), liver (FIG. 4D) and spleen (FIG. 4C) weredissected at 4 h after injection from the same mice in FIG. 3 . Tissueswere homogenized for enzyme assay. Data are expressed as mean±S.D.(n=4). The figure shows similar biodistribution, only significantdifference was lower GLA activity level in kidney for GLA produced inwt-CHO versus Fabrazyme.

FIG. 5 shows similar pharmacokinetic experiment as described in FIG. 3 .Five glycovariant GLA enzyme samples were compared with Fabrazyme.

FIG. 6 shows similar biodistribution experiment as described in FIG. 4 .Tissue distribution of the five glycovariant GLA enzyme samples werecompared with Fabrazyme. Heart (FIG. 6A), kidney (FIG. 6C), liver (FIG.6D) and spleen (FIG. 6B) were dissected at 24 h after injection from thesame mice in FIG. 5 .

FIG. 7 shows relative distribution of GLA variants into thedifficult-to-reach organs heart and kidney. GLA activities per wholeorgans were calculated. The sum of activities recovered from the 4organs (FIG. 4 ) was taken as total activity, and ratio recovered inheart and kidney was calculated. The data shows that compared toFabrazyme a higher proportion of the GLA-bi23SA reaches heart and kidneyand a lower proportion goes to the liver.

FIG. 8 shows the clearance of Gb3 substrate in tissues of Fabry mice.Residual Gb3 contents in liver (FIG. 8A), kidney (FIG. 8B), and heart(FIG. 8C) was analyzed 2 weeks after a single injection of 1 mg/kg ofGLA-bi23SA or Fabrazyme into 6 months old female Fabry mice viatail-vein. Data are presented as mean±S.D. (n=5). Statisticalsignificance shown on top of Fabrazyme and GLA-bi23SA-injected groupsindicates difference with vehicle alone (saline) treated Fabry mice.*P<0.05, **P<0.01, ***P<0.001.

FIG. 9 shows immunohistochemistry showing the cellular distribution ofinfused enzymes in liver (FIG. 9A), kidney (FIG. 9B), and heart (FIG.9C) of Fabry mice. Annotation in liver IHC: hepatocytes (small arrows),putative Kupffer cells (arrowheads), endothelial cells of sinusoidalcapillaries (large arrows) and punctate lysosome-like distribution ofpositive signals (small arrows); Annotation in kidney IHC: corticaltubules (indicated as ‘T’), glomeruli (indicated as ‘G’), and tubularepithelial cells (arrows); Annotation in heart IHC: vascular andperivascular cells (arrows).

FIG. 10 shows site specific glycoanalysis of GBA expressed in wt CHOcells or an engineered cell line with knock-out of Gnptab. The two mostabundant glycan structures at each of the four N-glycosites N19, N59,N146 and N270 of GBA are shown.

FIG. 11 shows a graphic depiction of gene targeting screen performed inCHO cells with general trend effects on N-glycosylation of GLA.CRISPR/Cas9 targeted genes are indicated with their predicted functions.The general trend effects of KO targeting are indicated for changes intotal sialic acid capping (SA), M6P-tagging (M6P), and exposed terminalmannose (Man) with arrows indicating increase/decrease. Glycan symbolsaccording to SNFG format (Varki, A. et al. Symbol Nomenclature forGraphical Representations of Glycans. Glycobiology 25, 1323-1324 (2015),incorporated herein by reference in its entirety).

FIG. 12 shows site-specific glycan analyses of selected genes targetedin the initial KO screen. The two most abundant glycan structures atN-glycosites N108, N161 and N184 of GLA produced in CHO WT (a) and CHOKO clones (b-t) are shown. Additional KO targeting and a detailedN-glycan analyses are shown in FIG. 17 . Each glycan structure wasconfirmed by targeted MS/MS analysis (FIG. 20 ). Details regarding thestacking ancestry, sequence analysis are shown in Supplementary Tables 2and 3.

FIG. 13 shows site-specific glycan analyses of combinatorial geneengineering. The two most abundant glycan structures at N-glycositesN108, N161 and N184 of GLA produced in KO/KI engineered CHO clones areshown (a-1). Each glyco structure was confirmed by targeted MS/MSanalysis. Additional KO targeting and a detailed N-glycan analyses areshown in FIG. 17 . Each glyco structure was confirmed by targeted MS/MSanalysis. Details regarding the stacking ancestry, sequence analysis areshown in Tables 6 and 7.

FIG. 14 shows tests demonstrating the universality of glycoengineeringusing recombinant GBA as reporter glycoprotein. The two most abundantglycan structures at N-glycosites N19, N59, N146 and N270 of GBAproduced CHO WT (a) and CHO KO clones (b-h) are shown. Each glycostructure was confirmed by targeted MS/MS analysis. Details regardingthe stacking ancestry, sequence analysis and N-glycans profiling areshown in Tables 8 and 9, and FIG. 19 , respectively.

FIG. 15 shows an in vivo study of the pharmacokinetics, biodistributionand Gb3 clearance of different GLA glycovariants in Fabry mice. FIG. 15Ashows a summary of the glycan features of GLA glycovariants used. Thetwo most abundant N-glycans are illustrated and detailed structuresshown in FIG. 17 , Panels 1, 6, 55, 52, 59 and 58). FIGS. 15B-15C showtime-courses of plasma GLA activities expressed as % of activity at 5min after injection (n=4 except for Fabrazyme in panel c where n=3).FIGS. 15D-15E show GLA enzyme activity in organs dissected afterinjection (same groups used as in FIGS. 15B, 15C). FIG. 15F shows IHCanalysis with polyclonal anti-GLA antibody. Annotations used:liver—hepatocytes (small arrows), putative Kupffer cells (arrowheads),endothelial cells of sinusoidal capillaries (large arrows), and punctatelysosome-like distribution of positive signals (small arrows);kidney-cortical tubules (indicated as ‘T’), glomeruli (indicated as‘G’), and tubular epithelial cells (arrows); heart—vascular andperivascular cells (arrows). FIG. 15G shows Gb3 substrate levelsquantified by mass spectrometry in organs 2 weeks after a singleinfusion of 1 mg/kg. (n=5). *P<0.05, **P<0.01, ***P<0.001.

FIG. 16 shows RNAseq transcriptome profiling of CHO cells showingpredicted expression of selected genes. RNAseq analysis was performedwith CHO GS−/− cells as previously reported.³⁴ Genes known to functionin N-glycosylation and M6P-tagging, including glycosyltransferases,glycosylhydrolases, enzymes involved in dolichol-linked precursoroligosaccharide synthesis, and other related genes, are shown.

FIG. 17 shows detailed presentation of N-glycans identified bysite-specific N-glycan profiling of GLA produced in CHO WT andengineered KO/KI clones as indicated. N-glycan structures and theirrelative abundances at each of the three N-glycosites (N108, N161, andN184) of GLA are illustrated with their relative abundance adjusted tothe most abundant structure. Minor glycoforms identified with relativeabundance less than 10% are not shown. Same N-glycan composition mayrepresent isobaric structures.

FIG. 18 shows characterization of growth and yield performance ofglycoengineered CHO clones expressing GLA. FIG. 18A shows viable celldensity and FIG. 18B shows cell viability as determined by Trypan blueexclusion test. Data from day 1-5 are shown as accurate cell countingafter day 6 was complicated by tendency for clumping of cells.

FIG. 18C shows yield of GLA enzyme activity determined in culture medium(2.5 μL) by release of p-nitrophenol per hour with a pNP-Gal enzymeassay. The substrate concentration was reduced to 1.2 mM to fit thelinear regression of absorbance at 405 nm FIG. 18D shows SDS-PAGECoomassie analysis of GLA in culture medium (10 μL loaded) after two,five and eight days of culture (D2, D5 and D8, respectively).

FIG. 19 shows detailed presentation of N-glycans identified bysite-specific N-glycan profiling of GBA produced in CHO WT andengineered KO clones as indicated. N-glycan structures and theirrelative abundances at each of the four N-glycosites (N19, N59, N146,and N270) of GBA are illustrated with their relative abundance adjustedto the most abundant structure. Minor glycoforms identified withrelative abundance less than 10% are not shown. Same N-glycancomposition may represent isobaric structures.

FIG. 20 shows targeted MS/MS manual annotation of the mostrepresentative glycopeptides. FIG. 20A shows extracted ion chromatogramsof 5 representative glycopeptide precursors of GLA produced in CHO WT.FIGS. 20B-20F show MS2 manual annotation of these precursors.

FIG. 21 shows in vitro assays of enzyme specific activity and plasmastability of GLA glycovariants. FIG. 21A shows specific activity of GLAvariants assessed by a pNP-Gal enzyme assay. Release of p-nitrophenolper hour was used to show the enzyme activity. Each data pointrepresents the mean of three replicates f SD. FIG. 21B shows in vitrostability of GLA variants (10 μg/ml in mice plasma) heated at 37° C. wasmeasured by the remaining activity. Each data point represents the meanof three replicates SD.

FIG. 22 shows relative distribution of GLA glycovariants among the fourmajor visceral organs: liver, spleen, kidney, and heart.

FIG. 23 shows similar biodistribution experiment as described in FIG. 22Tissue distribution of the GLA-bi23SA enzyme sample was compared withFabrazyme. Enzyme preparations were injected into Fabry mice at a doseof 0.2 or 0.5 mg/kg. Heart, kidney, and liver were dissected one weekafter injection and enzyme activity in the organ lysates was determined.

FIG. 24 shows the clearance of Gb3 substrate in tissues of Fabry mice.Residual Gb3 contents in kidney and heart was analyzed one weeks after asingle injection of 0.5 or 0.2 mg/kg of GLA-bi23SA or Fabrazyme into 6months old female Fabry mice via tail-vein. Data are presented as mean:S.D. (n=5). NS P>0.05.

FIG. 25 shows the clearance of Gb3 substrate in tissues of Fabry mice.Residual Gb3 contents in kidney, liver, and heart was analyzed 2 weeksafter a single injection of 1.0 mg/kg of GLA-26SA, GLA-Bi23SA orFabrazyme into 6 months old female Fabry mice via tail-vein. Data arepresented as mean±S.D. (n=5). NS P>0.05.

FIG. 26 shows enzyme activity in plasma at various time points afterinjection of GLA. Fabry rats were injected with 1 mg/kg dose of wt-CHOproduced GLA (Control) or GLA-bi23SA via tail vein (n=3 for GLA-bi23SAand n=1 for control group, male mice, age 12-14 weeks). At indicatedtime points, small amount of blood samples was collected from tail veinand separated plasma was analyzed for GLA activity. Enzyme activitieswere shown as % of activity at 5 min after injection. For GLA-bi23SA thedata are expressed as mean values (n=3). Solid lines are rat data anddashed lines are mouse data. The figure shows prolonged plasma clearancecurves for GLA-bi23SA versus control GLA. For comparison the mouse datapreviously obtained for GLA-bi23SA and control enzyme (Example 3, FIG.15 ) are reproduced from Tian et al. 2019 (average of n=5 per mousegroup). For GLA-bi23SA and control GLA the plasma clearance curves inthe rat are very similar to mouse curves, demonstrating same prolongedkinetics in rat and mouse.

FIG. 27 shows enzyme activity in balb/c mice injected with 0.75 mg/kg ofwild-type enzyme or optimized glycoengineered variant (-opt). For eachvariant blood samples were drawn at 30 and 120 min for AGA (FIG. 27B),GUSB (FIG. 27C) and Laman (FIG. 27C) enzyme and at 15 and 30 min for GLA(FIG. 27A) enzyme. Optimized enzyme variants are presented as solidboxes and wild-type enzyme is open boxes. The enzyme activity ispresented as percent of the initial plasma activity.

DETAILED DISCLOSURE OF THE INVENTION

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of molecular biology,recombinant DNA techniques, protein expression, andprotein/peptide/carbohydrate chemistry within the skill of the art, manyof which are described below for the purpose of illustration. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2000); DNACloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984); OligonucleotideSynthesis: Methods and Applications (P. Herdewijn, ed., 2004); NucleicAcid Hybridization (B. Hames & S. Higgins, eds., 1985); Nucleic AcidHybridization: Modem Applications (Buzdin and Lukyanov, eds., 2009);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Freshney, R. I. (2005)Culture of Animal Cells, a Manual of Basic Technique, 5th Ed. HobokenN.J., John Wiley & Sons; B. Perbal, A Practical Guide to MolecularCloning (3rd Edition 2010); Farrell, R., RNA Methodologies: A LaboratoryGuide for Isolation and Characterization (3rd Edition 2005).Poly(ethylene glycol), Chemistry and Biological Applications, ACS,Wash., 1997; Veronese, F., and J. M. Harris, Eds., Peptide and proteinPEGylation, Advanced Drug Delivery Reviews, 54(4) 453-609 (2002);Zalipsky, S., et al., “Use of functionalized Poly(Ethylene Glycols) formodification of polypeptides” in Polyethylene Glycol Chemistry:Biotechnical and Biomedical Applications. The publications discussedabove are provided solely for their disclosure before the filing date ofthe present application. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention. It is an object of the present inventionto provide novel glycoforms for lysosomal enzymes, resulting in modifiedbiodistribution of enzyme replacement therapies and more efficienttreatment of lysosomal storage disease.

Definitions

The articles “a” and “an” are used in this disclosure to refer to one ormore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “and/or” is used in this disclosure to mean either “and” or“or” unless indicated otherwise.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length. In any embodiment discussed in the context ofa numerical value used in conjunction with the term “about,” it isspecifically contemplated that the term about can be omitted.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises,” and “comprising” will be understoodto imply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that other elementsare optional and may or may not be present depending upon whether or notthey materially affect the activity or action of the listed elements.

Reference to the term “e.g.” is intended to mean “e.g., but not limitedto” and thus it should be understood that whatever follows is merely anexample of a particular embodiment, but should in no way be construed asbeing a limiting example. Unless otherwise indicated, use of “e.g.” isintended to explicitly indicate that other embodiments have beencontemplated and are encompassed by the present invention.

Reference throughout this specification to “embodiment” or “oneembodiment” or “an embodiment” or “some embodiments” or “certainembodiments” means that a particular feature, structure orcharacteristic described in connection with the embodiment is includedin at least one embodiment of the present invention. Thus, theappearances of the phrases “in one embodiment” or “in an embodiment” or“in certain embodiments” in various places throughout this specificationare not necessarily all referring to the same embodiment. Furthermore,the particular features, structures, or characteristics may be combinedin any suitable manner in one or more embodiments.

An “increased” or “enhanced” amount is typically a “statisticallysignificant” amount, and may include an increase that is 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times)(including all integers and decimal points in between and above 1, e.g.,2.1, 2.2, 2.3, 2.4, etc.) an amount or level described herein.Similarly, a “decreased” or “reduced” or “lesser” amount is typically a“statistically significant” amount, and may include a decrease that isabout 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4,4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100,500, 1000 times) (including all integers and decimal points in betweenand above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) an amount or leveldescribed herein.

“Optional” or “optionally” means that the subsequently described event,or circumstances, may or may not occur and that the description includesinstances where said event or circumstance occurs and instances in whichit does not.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95% or greater of some given quantity.

A “subject,” as used herein, includes any animal that exhibits asymptom, or is at risk for exhibiting a symptom, which can be treatedwith a composition or method disclosed herein. Suitable subjects(patients) include laboratory animals (such as mouse, rat, rabbit, orguinea pig), farm animals, and domestic animals or pets (such as a cator dog). Non-human primates and, preferably, human patients, areincluded.

“Treatment” or “treating,” as used herein, includes any desirable effecton the symptoms or pathology of a disease or condition, and may includeeven minimal changes or improvements in one or more measurable markersof the disease or condition being treated. “Treatment” or “treating”does not necessarily indicate complete eradication or cure of thedisease or condition, or associated symptoms thereof. The subjectreceiving this treatment is any subject in need thereof. Exemplarymarkers of clinical improvement will be apparent to persons skilled inthe art.

“Therapeutic response” refers to improvement of symptoms (whether or notsustained) based on the administration of the therapeutic response(whether or not tolerance is induced).

As used herein, the terms “therapeutically effective amount”,“therapeutic dose”, is the amount of the drug, e.g., a modifiedlysosomal enzyme described herein, needed to elicit the desiredbiological response following administration.

The term “administer”, “administering”, or “administration” as used inthis disclosure refers to either directly administering a disclosedcompound or pharmaceutically acceptable salt of the disclosed compoundor a composition to a subject, or administering a prodrug derivative oranalog of the compound or pharmaceutically acceptable salt of thecompound or composition to the subject, which can form an equivalentamount of active compound within the subject's body.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, excipients, ionicstrength modifiers, surfactants, buffers, carrier solutions,suspensions, colloids, and the like. The use of such media and agentsfor pharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g., a foreign gene) can beintroduced into a host cell so as to transform the host and promoteexpression (e.g., transcription and translation) of the introducedsequence. Vectors may include plasmids, phages, viruses, etc. and areknown in the art and discussed in greater detail below.

“Lysosomal Storage Disease” refers to inherited metabolic disorders thatresult from defects in lysosomal function, usually as a consequence ofdeficiency of a single lysosomal enzyme.

“Enzyme Replacement Therapy” refers to treatment by administration ofexogenous enzyme to compensate for the enzyme activity that is deficientin a given lysosomal storage disease.

The terms “Difficult-to-reach organ” and “hard-to-reach organ” are usedinterchangeably to refer to organs that are poorly treated with theexisting ERT's, including brain, kidney, heart and muscle and/orspecific cell types in these organs.

“N-glycosylation” refers to the attachment of the sugar moleculeoligosaccharide known as glycan to a nitrogen atom residue of a protein

“Sialylation” is the enzymatic addition of a neuraminic acid residue.

“High” sialic acid capping is used herein to refer to more than 4 molesof sialic acid per mole of enzyme. So, for example, a lysosomal enzymedisclosed herein that is said to have “high 2,3 sialic acid capping” isto be understood to be a lysosomal enzyme with more than 4 sialic acidmoieties per mole of enzyme, wherein the sialic acid moieties areattached via 2,3, sialyation.

“Neuraminic acid” (or NeuAc) is a 9-carbon monosaccharide, a derivativeof a ketononose.

“Biantennary” N-linked glycan is the simplest of the complex N-linkedglycans consisting of the N-glycan core(Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser/Thr) elongated withtwo GlcNAc residues linked to C-2 and of the mannose α1-3 and themannose α1-6. This core structure can then be elongated or modified byvarious glycan structures.

“Tri-antennary” N-linked glycans are formed when an additional GlcNAcresidue is added to either the C-4 of the core mannose α1-3 or the C-6of the core mannose α1-6 of the bi-antennary core structure. Thisstructure can then be elongated or modified by various glycanstructures.

“Tetra-antennary” N-linked glycans are formed when two additional GlcNAcresidues are added to either the C-4 of the core mannose α1-3 or the C-6of the core mannose α1-6 of the bi-antennary core structure. This corestructure can then be elongated or modified by various glycanstructures.

“Glycoprofiling” means characterization of glycan structures resident ona biological molecule or cell.

“Glycosyltransferases” are enzymes that catalyze the formation of theglycosidic linkage to form a glycoside. These enzymes utilize‘activated’ sugar phosphates as glycosyl donors, and catalyze glycosylgroup transfer to a nucleophilic group, usually an alcohol. The productof glycosyl transfer may be an O-, N-, S-, or C-glycoside; the glycosidemay be part of a monosaccharide, oligosaccharide, or polysaccharide.

“Glycogenes” includes glycosyltransferases and related glycogenes,wherein related glycogenes comprise any other enzyme acting on glycansto modify their structure. This included but is not limited tophosphotransferase, sulfotransferases, epimerases and deacetylases. Insome embodiments the related glycogene is a phosphotransferase orsubunit hereof.

“Glycosylation capacity” means the ability to produce an amount of aspecific glycan structure by a given cell or a given glycosylationprocess.

“Modified glycan profile” refers to change in number, type or positionof oligosaccharides in glycans on a given lysosomal enzyme.

In some embodiments, the term “mol” is used herein as an abbreviationfor “mole” (i.e., the unit of measurement for amount of substance in theInternational System of Units.

More “homogeneous glycosylation” means that the proportion of identicalglycan structures observed by glycoprofiling a given protein expressedin one cell is larger than the proportion ofidentical glycan structuresobserved by glycoprofiling the same protein expressed in another cell.

General Glycobiology—Basic glycobiology principles and definitions aredescribed in Varki et al. Essentials of Glycobiology, 3rd edition, 2017.

As used herein, reference to a gene identifier, regardless of whetherthe gene name is in all caps, lower case, or partial caps, refers to ahuman form of the gene unless context dictates otherwise. Thus, oneskilled in the art will understand that any reference to knocking-out aparticular gene will of course refer to knocking-out the endogenouslyexpressed gene in that organism unless explicitly stated otherwise. So,for example, although CHO cell genes are often annotated in the art withthe first letter capitalized and all remaining letters in lower case(e.g., St6gal1), whereas the human version of the same is oftenannotated in all caps (e.g., ST6GAL1), a reference herein toknocking-out “ST6GAL1” in CHO would mean that the endogenous CHO enzyme“St6gal1” was knocked out, even though the capitalization annotationmight have suggested a human enzyme was being knocked-out if stringentuse of such annotation were adhered to. Similarly, unless statedotherwise herein, reference to knocking-in “St6gal1” means the humanform of the gene has been knocked-in—not the CHO version. The sequencesof such genes are readily available on publically available databases,e.g., the NCBI database available at the world wide web address:ncbi.nlm.nih.gov/gene/, the content of which is incorporated herein byreference in its entirety.”

General DNA and molecular biology tools—Any of various techniques usedfor separating and recombining segments of DNA or genes, commonly by useof a restriction enzyme to cut a DNA fragment from donor DNA andinserting it into a plasmid or viral DNA. Using these techniques, DNAcoding for a protein of interest is recombined/cloned (using PCR and/orrestriction enzymes and DNA ligases or ligation independent methods suchas USER cloning) into a plasmid (known as an expression vector), whichcan subsequently be introduced into a cell by transfection using avariety of transfection methods such as calcium phosphate transfection,electroporation, microinjection and liposome transfection. Overview andsupplementary information and methods for constructing synthetic DNAsequences, insertion into plasmid vectors and subsequent transfectioninto cells can be found in Ausubel et al, 2003 and/or Sambrook &Russell, 2001.

“Gene” refers to a DNA region (including exons and introns) encoding agene product, as well as all DNA regions which regulate the productionof the gene product, whether or not such regulatory sequences areadjacent to coding and/or transcribed sequences or situated far awayfrom the gene which function they regulate. Accordingly, a geneincludes, but is not necessarily limited to, promoter sequences,terminators, translational regulatory sequences such as ribosome bindingsites and internal ribosome entry sites, enhancers, silencers,insulators, boundary elements, replication origins, matrix attachmentsites, and locus control regions. For homologous proteins the human androdent gene names are used inter-changeably (e.g. ST3GAL4, St3gal4).

“Targeted gene modifications”, “gene editing” or “genome editing” referto a process by which a specific chromosomal sequence is changed. Theedited chromosomal sequence may comprise an insertion of at least onenucleotide, a deletion of at least one nucleotide, and/or a substitutionof at least one nucleotide. Generally, genome editing inserts, replacesor removes nucleic acids from a genome using artificially engineerednucleases such as Zinc finger nucleases (ZFNs), TranscriptionActivator-Like Effector Nucleases (TALENs), the CRISPR/Cas system, andengineered meganuclease re-engineered homing endonucleases. Genomeediting principles are broadly used and thus known to person skilled inthe art.

“Endogenous” sequence/gene/protein refers to a chromosomal sequence orgene or protein that is native to the cell or originating from withinthe cell or organism analyzed.

“Exogenous” sequence or gene refers to a chromosomal sequence that isnot native to the cell, or a chromosomal sequence whose nativechromosomal location is in a different location in a chromosome ororiginating from outside the cell or organism analyzed.

“Heterologous” refers to an entity that is not native to the cell orspecies of interest.

The terms “nucleic acid” and “polynucleotide” refer to adeoxyribonucleotide or ribonucleotide polymer, in linear or circularconformation, and in either single- or double-stranded form. For thepurposes of the present disclosure, these terms are not to be construedas limiting with respect to the length of a polymer. The terms canencompass known analogs of natural nucleotides, as well as nucleotidesthat are modified in the base, sugar and/or phosphate moieties (e.g.,phosphorothioate backbones). In general, an analog of a particularnucleotide has the same base-pairing specificity; i.e., an analog of Awill base-pair with T.

The term “nucleotide” refers to deoxyribonucleotides or ribonucleotides.The nucleotides may be standard nucleotides (i.e., adenosine, guanosine,cytidine, thymidine, and uridine) or nucleotide analogs. A nucleotideanalog refers to a nucleotide having a modified purine or pyrimidinebase or a modified ribose moiety. A nucleotide analog may be a naturallyoccurring nucleotide (e.g., inosine) or a non-naturally occurringnucleotide.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues. These terms may also refer toglycosylated variants of the “polypeptide” or “protein”, also termed“glycoprotein.” “Polypeptide”, “protein” and “glycoprotein” is usedinterchangeably throughout this disclosure.

The term “recombination” refers to a process of exchange of geneticinformation between two polynucleotides. For the purposes of thisdisclosure, “homologous recombination” refers to the specialized form ofsuch exchange that takes place, for example, during repair ofdouble-strand breaks in cells. This process requires sequence similaritybetween the two polynucleotides, uses a “donor” or “exchange” moleculeto template repair of a “target” molecule (i.e., the one thatexperienced the double-strand break), and is variously known as“non-crossover gene conversion” or “short tract gene conversion,”because it leads to the transfer of genetic information from the donorto the target. Without being bound by any particular theory, suchtransfer can involve mismatch correction of heteroduplex DNA that formsbetween the broken target and the donor, and/or “synthesis-dependentstrand annealing,” in which the donor is used to resynthesize geneticinformation that will become part of the target, and/or relatedprocesses. Such specialized homologous recombination often results in analteration of the sequence of the target molecule such that part or allof the sequence of the donor polynucleotide is incorporated into thetarget polynucleotide.

Sequence identity Techniques for determining nucleic acid and amino acidsequence identity are known in the art. Typically, such techniquesinclude determining the nucleotide sequence of the mRNA for a geneand/or determining the amino acid sequence encoded thereby, andcomparing these sequences to a second nucleotide or amino acid sequence.Genomic sequences can also be determined and compared in this fashion.In general, identity refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Two or more sequences(polynucleotide or amino acid) can be compared by determining theirpercent identity. The percent identity of two sequences, whether nucleicacid or amino acid sequences, is the number of exact matches between twoaligned sequences divided by the length of the shorter sequences andmultiplied by 100. An approximate alignment for nucleic acid sequencesis provided by the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2:482-489 (1981). This algorithm can beapplied to amino acid sequences by using the scoring matrix developed byDayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5suppl. 3:353-358, National Biomedical Research Foundation, Washington,D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763(1986). An exemplary implementation of this algorithm to determinepercent identity of a sequence is provided by the Genetics ComputerGroup (Madison, Wis.) in the “BestFit” utility application. Othersuitable programs for calculating the percent identity or similaritybetween sequences are generally known in the art, for example, anotheralignment program is BLAST, used with default parameters. For example,BLASTN and BLASTP can be used using the following default parameters:genetic code=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+Swiss protein+Spupdate+PIR. Details of these programs canbe found on the GenBank website. With respect to sequences describedherein, the range of desired degrees of sequence identity isapproximately 80% to 100% and any integer value therebetween. Typicallythe percent identities between sequences are at least 70-75%, preferably80-82%, more preferably 85-90%, even more preferably 92%, still morepreferably 95%, and most preferably 98% sequence identity.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Although any methods,compositions, reagents, cells, similar or equivalent to those describedherein can be used in the practice or testing of the invention, thepreferred methods and materials are described herein. All publicationsand references, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference in their entirety as if each individual publication orreference were specifically and individually indicated to beincorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

Overview

The present disclosure provides novel glycoengineered lysosomal enzymes,and recombinant host cells for the homogeneous in vitro production ofthe same, for producing improved enzyme replacement therapy forlysosomal storage diseases including Fabry disease. In some embodiments,the novel glycoengineered lysosomal enzymes are produced in recombinanthost cells engineered according to the present disclosure to be capableof producing homogeneous glycoforms of lysosomal enzyme, e.g., via thegenetic knock-in and/or knock-out of one or more glycosyltransferasegenes.

In some embodiments, any lysosomal enzyme may be glycoengineeredaccording to the present disclosure. For example, in some embodiments,the lysosomal enzymes described herein that may be engineered accordingto the present disclosure include, human glycocerebrosidase (GC),iduronate 2-sulfatase (IDS), human arylsulfatase B(N-acetylgalactosamine-4-sulfatase) (ARSB), human lysosomalα-glucosidase (GAA), human alpha-galactosidase (GLA), humanbeta-glucuronidase (GUSB), human alpha-L-iduronidase (IDUA), humaniduronate 2-sulfatase (IDS), human beta-hexosaminidase alpha (HEXA),human beta-hexosaminidase beta (HEXB), human lysosomal α-mannosidase(mannosidase alpha class 2B member 1) (MAN2B1), human glucosylceramidase(GBA), human lysosomal acid lipase/cholesteryl ester hydrolase (lipaseA, lysosomal acid type)(LIPA), human aspartylglucosaminidase(N(4)-(beta-N-acetylglucosaminyl)-L-asparaginase) (AGA), humangalactosylceramidase (GALC)., human alpha-sulfatases, humanglucuronidase, human tripeptidyl peptidase 1 (TPP1), and humaniduronidase.

In some aspects, the present disclosure provides novel GLA compoundsallowing development of improved enzyme replacement therapy for Fabrydisease.

In some aspects, the present disclosure provides lysosomal enzymes withlow or no exposed mannose-6-phosphate (Man6P). In some aspects, “low”exposed Man6P means less 0.3 moles of exposed Man6P/mol enzyme. Suchenzymes may display increased circulation time in plasma, e.g., due todecreased interaction with mannose-6-phosphate receptors.

In some aspects, the present disclosure provides lysosomal enzymes withand/or low or no exposed mannose (Man). Such enzymes may displayincreased circulation time in plasma, e.g., due to decreased interactionwith mannose receptors.

In some aspects, the present disclosure provides lysosomal enzymes withhigh sialic acid capping and low or no exposed mannose-6-phosphate(Man6P) and/or low or no exposed mannose (Man). Such enzymes may displayincreased circulation time in plasma, e.g., due to decreased interactionwith mannose or mannose-6-phosphate receptors. In certain particularaspects, the sialic acid capping is via 2,3 linkage, but not 2,6linkage. In certain particular aspects, lysosomal enzymes engineered tohave terminal 2,3 sialylation and low or no mannose-6-phosphate (Man6P)and/or low or no mannose (Man). In some aspects, such enzymes display(i) improved or maintained biodistribution to difficult-to-reach organs,(ii) unchanged or prolonged serum half-life, (iii) unchanged orprolonged blood clearance, and/or (iv) maintained or increased enzymeactivity in plasma and/or in difficult-to-reach organs as compared tothe same lysosomal enzymes lacking such engineering.

In some aspects, the present disclosure provides lysosomal enzymes withlow Man6P and high sialic acid capping of alpha2,3 type.

In some aspects, the present disclosure provides mammalian cells (e.g.,a CHO cell) with glycosylation capacities engineered to producelysosomal enzymes with substantially altered content of Man6P, exposedMan, and/or content of sialic acid. Such cells may contain, e.g., one ormore inactivation of an endogenous glycogene and/or one or moreintroduction of an exogenous glycogene. In some aspects, the glycogenesmay be selected from the group consisting of GNPTAB, GNPTG, NAGPA, ALG3,ALG5, ALG6, ALG8, ALG9, ALG10, ALG12, Mannosidases, MAN1A1, MAN1A2,MAN1B1, MAN1C1, MAN2A1, MAN2A2, MOGS, GANAB, MGAT1, MGAT2 and Sialyltransferases. For example, the sialyl transferase may be St3gal4 andSt3gal6.

For example, in some non-limiting aspects, the mammalian cells (e.g.,CHO cells) may comprise a knockout of GNPTAB and/or a knockout of GNPTG.In some non-limiting aspects, the mammalian cells (e.g., CHO cells) maycomprise a knock-in of St3gal4 and/or St3gal6. In some non-limitingaspects, the mammalian cells (e.g., CHO cells) may comprise a knockoutof Mgat4b and/or a knockout of Mgat5. For example, in some non-limitingaspects, the mammalian cells (e.g., CHO cells) may comprise a knockoutof GNPTG and/or GNPTAB and a knock-in of St3gal4 and/or St3gal6. In somenon-limiting aspects, the mammalian cells (e.g., CHO cells) may comprisea knockout of one or more of GNPTG, GNPTAB, Mgat4b, Mgat5 and a knock-inof one or more of St3gal4 and St3gal6. For example, in one particularnon-limiting aspect, the mammalian cells (e.g., CHO cells) may comprisea knockout of GNPTG, GNPTAB, Mgat4b, Mgat5 and a knock-in of St3gal4. Inone particular non-limiting aspect, the mammalian cells (e.g., CHOcells) may comprise a knockout of GNPTG, GNPTAB, St3gal4, and St3gal6and a knock-in of St6gal1. In one particular non-limiting aspect, themammalian cells (e.g., CHO cells) may comprise a knockout of Alg3 and aknock-in of GNPTAB. In one particular non-limiting aspect, the mammaliancells (e.g., CHO cells) may comprise a knockout of ALG9. In oneparticular non-limiting aspect, the mammalian cells (e.g., CHO cells)may comprise a knock-in of GNPTAB and a knock-in of GNPTG.

In some embodiments of the present invention the mammalian cell isselected from the group consisting of HEK293 (e.g., HEK293-F, HEK293-H,HEK293-T, HEK293-6E), HT-1080, COS, VERO, MDCK, W138, V79, B14AF28-G3,CHO (e.g. CHO-K1, CHO-GS, CHO-S, CHO-ZN, CHO-DUKXB11, CHO-DG44), BHK,HaK, NS0, SP2/0-Ag14, HeLa, and PERC6 or derivatives of any of thesecells.

In some embodiments of the present invention is the mammalian cell aHEK293 cell.

In some embodiments of the present invention is the mammalian cell a CHOcell.

In some other embodiments of the present invention does theglycosylation of a lysosomal enzyme not comprise Man6P (for example byknock-out of Gnptg and/or Gnptab).

In some embodiments of the present invention the lysosomal enzymecontain less than 0.1 mol Man6P per mol enzyme, less than 0.2 mol Man6Pper mol enzyme, less than 0.3 mol Man6P per mol enzyme; less than 0.4mol Man6P per mol enzyme, less than 0.5 mol Man6P per mol enzyme.

In some embodiments of the present invention the lysosomal enzymecontains more than 7 mol alpha2,3SA per mol enzyme, more than 6 molalpha2,3SA per mol enzyme, more than 5 mol alpha2,3SA per mol enzyme,more than 4.5 mol alpha2,3SA per mol enzyme, more than 4 mol alpha2,3SAper mol enzyme

In some embodiments of the present invention the lysosomal enzymecontains no M6P or exposed Man, but 4-5 moles of sialic acid per mole ofenzyme protein, or 5-6 moles of sialic acid per mole of enzyme, or 6-7moles of sialic acid per mole of enzyme, or more than 7 moles of sialicacid per mole of enzyme.

In some embodiments of the present invention the lysosomal enzymecontains no M6P, but 4-5 moles of sialic acid per mole of enzymeprotein, or 6-7 moles of sialic acid per mole of enzyme, or more than 7moles of sialic acid per mole of enzyme.

In some other embodiments of the present invention does the enzymeglycosylation comprise increased sialylation (for example by knock-in ofone or more of St6gal1, St3gal4 or St3gal6).

In some other embodiments of the present invention does theglycosylation comprise increased tri- and tetra-antennary structures(for example by knock-in of one or more of Mgat4A, Mgat4B or Mgat5).

In some other embodiments of the present invention does theglycosylation comprise homogeneous bi-antennary structures (for exampleby knock-out of one or more of Mgat4A, Mgat4B or Mgat5).

In some other embodiments of the present invention does the enzymeglycosylation comprise increased sialylation of alpha2,3 type (forexample by knock-in of St3gal4 and/or St3gal6).

In some other embodiments of the present invention does theglycosylation comprise increased sialylation of homogeneous alpha2,3type (for example by knock-out of St6gal1 combined with knock-in ofSt3gal4/6) and no Man6P.

The present inventors have used different nuclease-mediated (ZFN, TALEN,CRISPR/Cas9) knock-out and knock-in in mammalian cells, such as HEK293or CHO cells, to obtain glycosylation capacities for producingglycovariants of lysosomal enzyme with improved circulation time and/orimproved targeting to diseased organs, like kidney, heart, muscle orbrain. As will be appreciated by the a person of average skill in theart, any location of DNA may be routinely targeted and cleaved using theZFN, TALEN, and CRISPR/Cas9 (and other Cas) systems enabling knock-outor knock-in of genes via methods well known in the art.

A zinc finger nuclease (ZFN) is an enzyme that is able to recognize andcleave a target nucleotide sequence with specificity due to the couplingof a “zinc finger DNA binding protein” (ZFP) (or binding domain), whichbinds DNA in a sequence-specific manner through one or more zincfingers, and a nuclease enzyme. ZFNs may comprise any suitable cleavagedomains (e.g., a nuclease enzyme) operatively linked to a ZFPDNA-binding domain to form a engineered ZFN that can facilitatesite-specific cleavage of a target DNA sequence (see, e.g., Kim et al.(1996) Proc Natl Acad Sci USA 93(3):1156-1160). For example, ZFNs maycomprise a target-specific ZFP linked to a FOK1 enzyme or a portion of aFOK1 enzyme. In some embodiments, ZFN used in a ZFN-mediated targetedintegration approach utilize two separate molecules, each comprising asubunit of a FOK1 enzyme each bound to a ZFP, each ZFP with specificityfor a DNA sequence flanking a target cleavage site, and when the twoZFPs bind to their respective target DNA sites the FOK1 enzyme subunitsare brought into proximity with one another and they bind togetheractivating the nuclease activity which cleaves the target cleavage site.ZFNs have been used for genome modification in a variety of organisms(e.g., United States Patent Publications 20030232410; 20050208489;20050026157; 20050064474; 20060188987; 20060063231; and InternationalPublication WO 07/014,275, incorporated herein by reference in theirentirety) Custom ZFPs and ZFNs are commercially available from, e.g.,Sigma Aldrich (St. Louis, Mo.), and any location of DNA may be routinelytargeted and cleaved using such custom ZFNs.

TALENS utilize a “TALE DNA binding domain” or “TALE,” which is apolypeptide comprising one or more TALE repeat domains/units. The repeatdomains are involved in binding of the TALE to its cognate target DNAsequence. A single “repeat unit” (also referred to as a “repeat”) istypically 33-35 amino acids in length and exhibits at least somesequence homology with other TALE repeat sequences within a naturallyoccurring TALE protein. TAL-effectors may contain a nuclear localizationsequence, an acidic transcriptional activation domain and a centralizeddomain of tandem repeats where each repeat contains approximately 34amino acids that are key to the DNA binding specificity of theseproteins. (e.g., Schornack S, et al (2006) J Plant Physiol 163(3):256-272). TAL effectors depend on the sequences found in the tandemrepeats which comprises approximately 102 bp and the repeats aretypically 91-100% homologous with each other (e.g., Bonas et al (1989)Mol Gen Genet 218: 127-136). These DNA binding repeats may be engineeredinto proteins with new combinations and numbers of repeats, to makeartificial transcription factors that are able to interact with newsequences and activate the expression of a non-endogenous reporter gene(e.g., Bonas et al (1989) MoI Gen Genet 218: 127-136). Engineered TALproteins may be linked to a FokI cleavage half domain to yield a TALeffector domain nuclease fusion (TALEN) to cleave target specific DNAsequence (e.g., Christian et al (2010) Genetics epub10.1534/genetics.110.120717).

Custom TALEN are commercially available from, e.g., Thermo FisherScientific (Waltham, Mass.), and any location of DNA may be routinelytargeted and cleaved.

Any location of DNA may be routinely targeted and cleaved using theCRISPR/Cas9 system enabling knock-out or knock-in of genes via methodswell known in the art.

A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas(CRISPR Associated) nuclease system is an engineered nuclease systembased on a bacterial system that may be used for genome engineering. Itis based on part of the adaptive immune response of many bacteria andarchaea. When a virus or plasmid invades a bacterium, segments of theinvader's DNA are converted into CRISPR RNAs (crRNA) by the ‘immune’response. This crRNA then associates, through a region of partialcomplementarity, with another type of RNA called tracrRNA to guide theCas9 nuclease to a region homologous to the crRNA in the target DNAcalled a “protospacer”. Cas9 cleaves the DNA to generate blunt ends atthe DSB at sites specified by a 20-nucleotide guide sequence containedwithin the crRNA transcript. Cas9 requires both the crRNA and thetracrRNA for site specific DNA recognition and cleavage. This system hasnow been engineered such that the crRNA and tracrRNA can be combinedinto one molecule (the “single guide RNA”), and the crRNA equivalentportion of the single guide RNA can be engineered to guide the Cas9nuclease to target any desired sequence (see Jinek et al (2012) Science337, p. 816-821, Jinek et al, (2013), eLife 2:e00471, and David Segal,(2013) eLife 2:e00563). Thus, the CRISPR/Cas system can be engineered tocreate a DSB at a desired target in a genome, and repair of the DSB canbe influenced by the use of repair inhibitors to cause an increase inerror prone repair. As will be clear to the skill artisan, other CRISPRnucleases, in addition to Cas9, are known and are suitable for use inthe present invention.

In some embodiments, the CRISPR/Cas nuclease-mediated integrationutilizes a Type II CRISPR. The Type II CRISPR is one of the most wellcharacterized systems and carries out targeted DNA double-strand breakin four sequential steps. First, two non-coding RNA, the pre-crRNA arrayand tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNAhybridizes to the repeat regions of the pre-crRNA and mediates theprocessing of pre-crRNA into mature crRNAs containing individual spacersequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to thetarget DNA via Watson-Crick base-pairing between the spacer on the crRNAand the protospacer on the target DNA next to a protospacer adjacentmotif (PAM), an additional requirement for target recognition. Forth,Cas9 mediates cleavage of target DNA to create a double-stranded breakwithin the protospacer.

The Cas9 related CRISPR/Cas system comprises two RNA non-codingcomponents: tracrRNA and a pre-crRNA array containing nuclease guidesequences (spacers) interspaced by identical direct repeats (DRs). Touse a CRISPR/Cas system to accomplish genome engineering, both functionsof these RNAs must be present (see Cong et al, (2013) Sciencexpress1/10.1126/science 1231143). In some embodiments, the tracrRNA andpre-crRNAs are supplied via separate expression constructs or asseparate RNAs. In other embodiments, a chimeric RNA is constructed wherean engineered mature crRNA (conferring target specificity) is fused to atracrRNA (supplying interaction with the Cas9) to create a chimericcr-RNA-tracrRNA hybrid (also termed a single guide RNA). (see Jinek ibidand Cong, ibid).

In some embodiments, a single guide RNA containing both the crRNA andtracrRNA may be engineered to guide the Cas9 nuclease to target anydesired sequence (e.g., Jinek et al (2012) Science 337, p. 816-821,Jinek et al, (2013), eLife 2:e00471, David Segal, (2013) eLife2:e00563). Thus, the CRISPR/Cas system may be engineered to create a DSBat a desired target in a genome.

Custom CRISPR/Cas systems are commercially available from, e.g.,Dharmacon (Lafayette, Colo.), and any location of DNA may be routinelytargeted and cleaved using such custom single guide RNA sequences.Single stranded DNA templates for recombination may be synthesized(e.g., via oligonucleotide synthesis methods known in the art andcommercially available) or provided in a vector, e.g., a viral vectorsuch as an AAV.

In some aspects, the present disclosure provides mammalian cells thatproduce lysosomal enzyme with one or more of the followingposttranslational modification patterns:

-   -   a) Elimination of Man6P on one or more N-glycans (for example by        knock-out of Gnptab and/or Gnptg),    -   b) Low Man6P on one or more N-glycans (for example by knock-out        of Gnptab and/or Gnptg),    -   c) High sialic acid content (>4 mol SA per mol enzyme) (for        example by knock-in of St3gal4/6),    -   d) no exposed mannose    -   e) Homogenous alpha2,3 SA capping (for example by knock-in of        one or more of St3gal4, and St3gal6)    -   f) Homogeneous biantennary glycans (for example by knock-out of        Mgat4A, Mgat4B and/or Mgat5), or    -   g) Higher number of antennae on glycans (for example by knock-in        of Mgat4A, Mgat4B and/or Mgat5)

One, two, three, four, five, six or seven of these effects can becombined to generate specific posttranslational modification patterns.

In some aspects of the present invention ST6GAL1 is knocked out in ahuman cell leading to homogeneous alpha2,3 sialylation of N-glycans.

In some aspects of the present invention Gnptab is knocked out in thecell leading to elimination of Man6P tagging of N-glycans, decrease inexposed-Man, and increase in sialylated N-glycans of lysosomal enzymeproteins.

In some aspects of the present invention Gnptg is knocked out in thecell leading to elimination of Man6P tagging of N-glycans, decrease inexposed-Man, and increase in sialylated N-glycans of lysosomal enzymeproteins.

In some aspects of the present invention GNPTAB and ST6GAL1 are knockedout and ST3GAL4 is knocked-in in the human cell leading to N-glycanswith alpha2,3-linked sialic acids and without Man6P tagging of N-glycansof lysosomal enzyme proteins.

In some aspects of the present invention Gnptab and ST3GAL4/6 areknocked out and ST6GAL1 is knocked in in the cell leading to complextype N-glycans with alpha2,6-linked sialic acids and without M6P taggingof N-glycans of lysosomal enzyme proteins.

In one aspect of the present invention is one or more of the abovementioned genes knocked out using zinc finger nucleases ZFN. ZFNs can beused for inactivation of any genes disclosed herein.

In one aspect of the present invention is one or more of the abovementioned genes knocked out using TALENs. TALENs can be used forinactivation of any genes disclosed herein.

In one aspect of the present invention is one or more of the abovementioned genes knocked out using CRISPR/Cas9. CRISPR/Cas9 can be usedfor inactivation of any genes disclosed herein.

In some embodiments, this disclosure provides mammalian cells withdifferent well-defined N-glycosylation capacities that enablerecombinant production of Lysosomal glycoprotein therapeutics withN-glycans with low Man6P tagging, with or without alpha2,6 SA capping,with or without alpha2,3 SA capping, and without exposed mannose.

In some aspects this disclosure provides an isolated cell comprising anyof the proteins and/or polynucleotides as described herein. In certainembodiments, one or more glycosyltransferase genes are inactivated(partially or fully) in the cell. Any of the cells described herein mayinclude additional genes that have been inactivated, for example, usingzinc finger nucleases, TALENs and/or CRISPR/Cas9 designed to bind to atarget sequence in the selected gene. In certain embodiments, providedherein are cells or mammalian cells in which two or moreglycosyltransferase genes have been inactivated, and cells or mammaliancells in which one or more glycosyltransferase and related glycogeneshave been inactivated and one or more glycosyltransferase genesintroduced.

In some embodiments, this invention provides a cell with inactivationand/or modification of the Man6P tagging process of N-glycans, and thatproduces lysosomal enzyme proteins with no or lower levels of Man6Ptagged N-glycans.

In some embodiments, introduction of one or more exogenousglycosyltransferase(s) is performed by plasmid transfection with aplasmid encoding constitutive promotor driven expression of both theglycosyltransferase gene and a selectable antibiotic marker, where theselectable marker could also represent an essential gene not present inthe host cell such as GS system (Sigma/Lonza), and/or separate plasmidsencoding the constitutive promotor driven glycosyltransferase gene orthe selectable marker. For example, plasmids encoding ST6GAL1 and Zeocinhave been transfected into cells and stable ST6GAL-I expressing lineshave been selected based on zeocin resistance

In some other embodiments, introduction of one or more exogenousglycosyltransferase(s) is performed by site-directed nuclease-mediatedinsertion.

In some aspects, the disclosure provides a method of producing arecombinant lysosomal enzyme of interest in a host cell, the methodcomprising the steps of: (a) providing a host cell comprising one ormore endogenous glycosyltransferase genes; (b) inactivating theendogenous glycosyltransferase gene(s) of the host cell by any of themethods described herein; and (c) introducing an expression vectorcomprising a transgene, the transgene comprising a sequence encoding alysosomal enzyme of interest, into the host cell, thereby producing therecombinant enzyme with low Man6P and low exposed Mannose and highsialic acid glycoforms.

In some aspects, the disclosure provides a method of producing arecombinant lysosomal enzyme of interest in a cell, the methodcomprising the steps of: (a) providing a cell comprising one or moreendogenous glycosyltransferase gene; (b) inactivating the endogenousglycosyltransferase gene(s) of the host cell; (c) introducing one ormore glycosyltransferase gene(s) in the cell by any of the methodsdescribed herein; and (d) introducing an expression vector comprising atransgene, the transgene comprising a sequence encoding a protein ofinterest, into the cell, thereby producing the recombinant lysosomalenzyme with low Man6P and low exposed mannose and high alpha2,3 typesialic acid glycoforms.

In some aspects, the disclosure provides a method of producing arecombinant lysosomal enzyme of interest in a cell, the methodcomprising the steps of: (a) introducing an expression vector comprisinga transgene, the transgene comprising a sequence encoding a lysosomalenzyme of interest, into the cell (b) isolating clonal cell lineproducing the lysosomal enzyme; (c) inactivating one or more of theendogenous glycogene(s) of the host cell; (c) introducing one or moreglycosyltransferase gene(s) in the cell by any of the methods describedherein; and (d), thereby producing the recombinant lysosomal enzyme withlow Man6P and low exposed mannose and high alpha2,3 type sialic acidglycoforms.

In any of the cells and methods described herein, the cell or cell linemay be a HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T, HEK293-6E),HT-1080, COS, VERO, MDCK, WI38, V79, B14AF28-G3, CHO (e.g. CHO-KI,CHO-GS, CHO-S, CHO-ZN, CHO-DUKXB11, CHO-DG44), BHK, HKB, HaK, NS0,SP2/0-Ag14, HeLa, and PERC6.

Knock-Out Targeting Strategy.

It is clear to the person skilled in the art that inactivation of a geneinvolved in glycosylation pathways can have a multitude of outcomes andeffects on the transcript and/or protein product translated from this.Targeted inactivation experiments performed herein involved PCR andsequencing of the introduced alterations in the genes as well as RNAseqanalysis of clones to determine whether a transcript was formed and ifpotential novel splice variations have possibly introduced new proteinstructures. Moreover, methods for determining presence of protein fromsuch transcripts are available and include mass spectrometry andSDS-PAGE Western blot analysis with relevant antibodies detecting themost N-terminal region of the protein products.

In some embodiments the cell may express an exogenous lysosomal enzyme,which lysosomal enzyme is expressed to comprise one or moreposttranslational modifications independently selected from:

-   -   a) with alpha2,3SA capping,    -   b) without alpha2,3SA capping,    -   c) with alpha2,6SA capping,    -   d) without alpha2,6SA capping,    -   e) without Man6P,    -   f) low Man6P,    -   g) without exposed Mannose,    -   h) low exposed Mannose,    -   i) >75% biantennary structures, and    -   j) >75% tri/tetra antennary structures.

In some embodiments the exogenous protein of interest is a lysosomalenzyme, and the one or more endogenous glycogene inactivated is Gnptabor Gnptg, such as in order to lower Man6P and increase sialic acids.

In some embodiments the exogenous protein of interest is a lysosomalenzyme, wherein said lysosomal enzyme has altered mannose-6-phosphate(Man6P) tagging of N-glycans and/or changed site occupancy of M6P, suchas by knocking out one or both of Gnptg and Gnptab genes.

In some embodiments of the present disclosure, glycosylation on alysosomal enzyme disclosed herein is made more homogenous.

In some other embodiments of the present disclosure, N glycans on alysosomal enzyme disclosed herein are sialylated with at least 7 mol SA,or at least 6, at least 5, at least 4.5, or at least 4 mol SA per mol ofenzyme.

In some other embodiments of the present disclosure N glycans on alysosomal enzyme disclosed herein are alpha2,3 sialylated with at least7 mol alpha2,3SA, or at least 6, at least 5, at least 4.5, or at least 4mol alpha2,3SA per mol of enzyme.

In some embodiment the modified lysosomal enzyme is fused to anon-glycan tag designed to improve blood-brain-barrier passage

In some embodiment the non-glycan tags fused to a modified lysosomalenzyme may include IGF2, Transferrin, Immunoglobin or any fragment orderivative of these

The optimal glycoform for a lysosomal enzyme may be identified by thefollowing process:

-   -   (i) producing the lysosomal enzyme in a panel of isogenic cells        with different glycosylation capacities, obtained by        inactivating and/or introducing one or more glycogenes in said        mammalian cells resulting in at least one novel glycosylation        capacity,    -   (ii) Optionally purify the produced glycovariants of the        lysosomal enzyme,    -   iii) determination of improved drug feature of each glycovariant        of the enzyme in suitable assay, e.g. in-vitro assays (binding        assay, enzyme assay, cell uptake assays) or animal studies        (Serum half-life, targeting to diseased organs, improved        biodistribution, substrate reduction). Improvement is based on        comparison with a reference enzyme in same assay; and    -   (iv) perform glycoanalysis for determination of the glycoform        with the improved drug feature

The above described process allows the identification of the optimalglycan structure for a particular lysosomal enzyme. For those skilled inthe art by using the genotype fingerprint identified in (iii) maygenerate an efficient engineered mammalian cell line with the optimalgenotype for efficient production of the lysosomal enzyme with saidglycan.

One aspect of the present disclosure relates to a method for producing alysosomal enzyme having modified glycan profile wherein the cellproducing the lysosomal enzyme has more than one modification of one ormore glycogenes.

In some embodiments of the present disclosure provides cells (e.g.,mammalian cells) that have been modified by glycogene knock-out and/orknock-in of an exogenous DNA sequence coding for a protein involved inglycosylation (e.g. a glycosyltransferase or a phospho transferase).

In some embodiments of the present disclosure a lysosomal enzyme isproduced in cells (e.g., mammalian cells) in which one or moreendogenous gene selected from the group consisting of ST3GAL4/6 andST6GAL1 have been knocked out.

In some embodiments of the present disclosure, a lysosomal enzyme isproduced in cells (e.g., mammalian cells) in which one or more exogenousgene selected from the group consisting of ST3GAL4, ST3GAL6, ST6GAL1,MGAT4A, MGAT 4B, MGAT5 have been knocked in.

In some embodiments of the present disclosure, a lysosomal enzyme isproduced in cells (e.g., mammalian cells) in which one or both of GNPTABor GNPTG have been knocked out

One aspect of the present disclosure relates to a method for producing alysosomal enzyme having a plurality of glycan profiles, and from thisplurality of glycovariant enzyme protein identifying glycovariants withimproved (drug) properties. The selection of such glycovariants withimproved (drug) properties may comprise analyzing the glycovariantenzyme for activity in comparison with a reference lysosomal enzyme in(a) suitable bioassay(s); and selection of the enzyme glycoform with thehigher/highest/optimal activity.

In one aspect of the present disclosure, one or more of the abovementioned genes knocked out using transcription activator-like effectornucleases (TALENs).

In one aspect of the present disclosure, one or more of the abovementioned genes is knocked out using CRISPRs (clustered regularlyinterspaced short palindromic repeats).

In one aspect of the present disclosure, one or more of the abovementioned genes knocked out using ZFN (Zinc Finger Nuclease).

In some embodiments of the present disclosure, the cell producing therecombinant lysosomal enzyme is selected from the group consisting ofCHO, HEK293, NS0, SP2/0, YB2/0, CAP, PERC6, HT-1080, NS0, SP2/0, and BHKcells.

In certain embodiments of the present disclosure the cell is a HEK293cell.

In certain embodiments of the present disclosure the cell is a CHO cell.

In some embodiments, the cell may be an isolated cell, a cell in cellculture, or a cell line.

In some embodiments the “naked” cells may be glycoengineered beforeexpressing the lysosomal enzyme of interest in the engineered cell line.

In some embodiments a cell line producing the recombinant lysosomalenzyme of interest is glycoengineered.

In some embodiments the glyco-engineering design comprising the specificknock-outs and knock-ins giving the optimal modified lysosomal enzymemay be transferred into a novel host cell line using any gene editingtechnology (ZFN, TALENs or CRISPR)

In some embodiments a CHO cell line producing an optimal human lysosomalenzyme glycovariant is engineered by way of gene knock-out to inactivatethe corresponding endogenous CHO lysosomal enzyme, so that only thehuman form of the enzyme is produced.

Due to short half-life of GLA enzyme in serum it has been co-formulatedwith 1-deoxygalactonojirimycin (DGJ) as disclosed in U.S. Pat. No.9,694,056 and published in Xu 2015. The DGJ increased the physicalstability, and increased the potency for GLA-mediatedglobotriaosylceramide reduction in cultured Fabry fibroblasts. In Fabrymice, co-formulation of 1-deoxygalactonojirimycin increased the totalexposure of active enzyme, and led to greater tissueglobotriaosylceramide reduction when compared with GLA alone.

In some embodiments, the present invention provides pharmaceuticalcompositions comprising one or more modified lysosomal enzyme disclosedherein. Such pharmaceutical compositions may comprise the one or moremodified lysosomal enzyme and one or more pharmaceutically acceptablecarrier, excipient, or vehicle.

The term “pharmaceutically acceptable carrier” includes any of thestandard pharmaceutical carriers. Pharmaceutically acceptable carriersfor therapeutic use are well known in the pharmaceutical art and aredescribed, for example, in “Remington's Pharmaceutical Sciences”, 17thedition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa.,USA, 1985. For example, sterile saline and phosphate-buffered saline atslightly acidic or physiological pH may be used. Suitable pH-bufferingagents may, e.g., be phosphate, citrate, acetate,tris(hydroxymethyl)aminomethane (TRIS),N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), ammoniumbicarbonate, diethanolamine, histidine, arginine, lysine or acetate(e.g. as sodium acetate), or mixtures thereof. The term furtherencompasses any carrier agents listed in the US Pharmacopeia for use inanimals, including humans.

A pharmaceutical composition of the invention may be in unit dosageform. In such form, the composition is divided into unit dosescontaining appropriate quantities of the active component or components.The unit dosage form may be presented as a packaged preparation, thepackage containing discrete quantities of the preparation, for example,packaged tablets, capsules or powders in vials or ampoules. The unitdosage form may also be, e.g., a capsule, cachet or tablet in itself, orit may be an appropriate number of any of these packaged forms. A unitdosage form may also be provided in single-dose injectable form, forexample in the form of a pen device containing a liquid-phase (typicallyaqueous) composition.

Pharmaceutical compositions comprising the one or more modifiedlysosomal enzyme may be formulated (e.g., with one or morepharmaceutically acceptable carrier, excipient, or diluent) for anysuitable route and means of administration. Pharmaceutically acceptablecarriers, excipiants or diluents include those used in formulationssuitable for e.g. oral, intravitreal, rectal, vaginal, nasal, topical,enteral or parenteral (including subcutaneous (SC), intramuscular (IM),intravenous (IV), intradermal and transdermal) administration oradministration by inhalation. The formulations may conveniently bepresented in unit dosage form and may be prepared by any of the methodswell known in the art of pharmaceutical formulation.

Subcutaneous or transdermal modes of administration may be particularlysuitable for the peptide analogues of the invention.

Further embodiments of the invention relate to devices, dosage forms andpackages used to deliver the pharmaceutical formulations of the presentinvention. Thus, at least one peptide analogue or specified portion orvariant in either the stable or preserved formulations or solutionsdescribed herein, can be administered to a patient in accordance withthe present invention via a variety of delivery methods, including SC orIM injection; transdermal, pulmonary, transmucosal, implant, osmoticpump, cartridge, micro pump, or other means appreciated by the skilledartisan as well-known in the art.

Still further embodiments of the invention may relate to oralformulations and oral administration. Formulations for oraladministration may rely on the co-administration of adjuvants (e.g.resorcinols and/or nonionic surfactants such as polyoxyethylene oleylether and n-hexadecylpolyethylene ether) to artificially increase thepermeability of the intestinal walls, and/or the co-administration ofenzymatic inhibitors (e.g. pancreatic trypsin inhibitors,diisopropylfluorophosphate (DFF) or trasylol) to inhibit enzymaticdegradation. The active constituent compound of a solid-type dosage formfor oral administration can be mixed with at least one additive, such assucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol,dextran, starches, agar, alginates, chitins, chitosans, pectins, gumtragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic orsemisynthetic polymer, or glyceride. These dosage forms can also containother type(s) of additives, e.g., inactive diluting agent, lubricantsuch as magnesium stearate, paraben, preserving agent such as sorbicacid, ascorbic acid, alpha-tocopherol, antioxidants such as cysteine,disintegrators, binders, thickeners, buffering agents, pH adjustingagents, sweetening agents, flavoring agents or perfuming agents.

The components used to formulate the pharmaceutical compositions arepreferably of high purity and are substantially free of potentiallyharmful contaminants (e.g., at least National Food (NF) grade, generallyat least analytical grade, and more typically at least pharmaceuticalgrade). Moreover, compositions intended for in vivo use are usuallysterile. To the extent that a given compound must be synthesized priorto use, the resulting product is typically substantially free of anypotentially toxic agents, particularly any endotoxins, which may bepresent during the synthesis or purification process. Compositions forparental administration are also sterile, substantially isotonic andmade under GMP conditions.

In some embodiments, the invention relates to a kit comprising one ormore modified lysosomal enzyme of the invention. In other embodiments,the kit comprises one or more pharmaceutical compositions comprising oneor more modified lysosomal enzyme. In certain embodiments, the kitfurther comprises packaging or instructions for use.

In some embodiments the modified lysosomal enzymes disclosed herein maybe co-formulated with a compound that improves the serum stability ofthe enzyme. In some embodiments the modified lysosomal enzyme mayco-formulated with DGJ. The present disclosure also provides for the useof a co-formulation of modified α-Gal A and DGJ or a pharmaceuticallyacceptable salt thereof in the preparation of a medicament for thetreatment of Fabry disease, wherein the medicament is formulated forparenteral administration to a subject, and wherein the α-Gal A of theco-formulation is formulated for administration in an amount of about0.3, 0.5, 1, 2 or 3 mg/kg, and the DGJ of the co-formulation isformulated for administration in an amount of about 0.1, 0.3, 0.5, 1, 3,or 10 mg/kg.

In some embodiments the lysosomal enzyme modifications or glycodesignsdisclosed herein (e.g., any one or more of the modified GLA enzymesdisclosed herein) may be combined with enzyme stabilizing technologiesinvolving amino acid mutations in the enzyme polypeptide sequence toobtain additive or synergistic effects. In some embodiments, suchmutated enzymes have improved thermal and/or physical stability and/orimproved cell uptake properties.

It should be noted that embodiments and features described in thecontext of one of the aspects or embodiments of the present inventionalso apply to the other aspects and embodiments of the invention.

All patent and non-patent references cited in the present application,are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the followingnon-limiting examples.

EXAMPLES

The following examples are given as an illustration of variousembodiments of the invention and are thus not meant to limit the presentinvention in any way. Along with the present examples the methodsdescribed herein are exemplary, and are not intended as limitations onthe scope of the invention. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

Example 1—Production and Characterization of Human GLA Enzyme

The alpha-galactosidase A (GLA) enzyme is 398 amino acids glycoproteinwith three N-glycan sites. Lack of GLA enzyme activity is cause of thelysosomal disease called Fabry's disease. There are two replacementenzyme products available for Fabry disease and we used the Fabrazymefrom Genzyme/Sanofi as control for our experiments. For producingrecombinant human alpha-galactosidase A (GLA) we first generated astable high expressing CHO cell line.

Generation of GLA Production Cell Line:

An expression construct containing the entire coding sequence of humanGLA was synthesized by Genewiz (USA) and subcloned into modified pCGS3(Merck/Sigma-Aldrich) for glutamine selection in CHOZN GS−/− cells. CHOcells were maintained as suspension cultures in EX-CELL CD CHO Fusionserum-free media (Sigma), supplemented with 4 mM L-glutamine in 50 mLTPP TubeSpin@ Bioreactors with 180 rpm shaking speed at 37° C. and 5%CO2. Cells were seeded at 0.5 mil cells/mL in T25 flask (NUNC) one dayprior to transfection. Electroporation was conducted with 2 mil cellsand 8 μg endotoxin-free plasmids using Amaxa kit V and program U24 withAmaxa Nucleofector 2B (Lonza, Switzerland). Electroporated cells wereplated in 6-well plates with 3 mL growth medium, and after 72 h cellswere transferred to 96w plates at 1,000 cells/well in 200 μl MinipoolPlating Medium containing 80% EX CELL@ CHO Cloning Medium (Cat.no C6366)and EX-CELL CHO CD Fusion serum-free medium without glutamine. Highexpressing clones were selected by GLA enzyme activity in the medium.

For producing GLA enzyme the CHO cell line was cultured in EX-CELL CHOCD serum-free media without L-glutamine in 50 mL TPP TubeSpin®Bioreactors or Erlenmeyer flasks and incubated with shaking (180 rpm,37° C. and 5% CO₂) for 4 days Spent culture medium was collected aftercentrifugation at 500×g for 20 min, filtered through 0.45 μm filter,diluted 3-fold with 25 mM MES (pH 6.0), and loaded onto a DEAE-SepharoseFast Flow column (Sigma). The column was washed with 10 column volumes(CV) washing buffer (25 mM MES with 50 mM NaCl, pH 6.0) and eluted with5 CV elution buffer (25 mM MES with 200 mM NaCl, pH 6.0). For GBA theHis-tagged protein was purified by nickel affinity purification(Invitrogen, US). Culture medium was centrifuged, filtered, and mixed3:1 (v/v) in 4× binding buffer (200 mM Tris, pH 8.0, 1.2 M NaCl) andapplied to 0.3 ml packed NiNTA agarose (Invitrogen), pre-equilibrated inbinding buffer (50 mM Tris, pH 8.0, 300 mM NaCl). The column was washedwith binding buffer and then bound protein was eluted with bindingbuffer with an additional 250 mM imidazole. Purity and quantity wasevaluated by SDS-PAGE Coomassie staining and enzyme activity wasanalyzed using GLA activity assay.

GLA Activity Assay:

GLA enzyme activity was measured with 33 mMp-nitrophenyl-α-D-galactopyranoside (pNP-Gal) substrate at 37° C. for 30min at pH 4.6 in 20 mM citrate and 30 mM sodium phosphate, and thereaction was quenched with borate buffer (pH 9.8) and releasedp-nitrophenol was read at 405 nm.

Site Specific Glycoanalysis of GLA Enzyme:

To characterize the glycans of the GLA enzyme we used site-specificglycoprofiling of the secreted GLA. GLA has three N-glycosites (N108,N161, N184) and peptide digestion with chymotrypsin was used to isolatethe corresponding glycopeptides. Approximately 10 μg of purified GLA in50 mM Ammoniumbicarbonate buffer (pH 7.4) was reduced withdithiothreitol (10 mM) at 60° C. for 30 min and alkylated withiodoacetamide (20 mM) for 30 min in dark at room temperature.Chymotrypsin digestion was performed at a 1:25 enzyme: substrate ratio.The proteolytic digest was desalted by custom made modified StageTipcolumns containing 2 layers of C18 and 1 layer of C8 membrane (3M Emporedisks, Sigma-Aldrich). Samples were eluted with 50% methanol in 0.1%formic acid, and then dried in SpeedVac and re-solubilized in 0.1%formic acid. LC MS/MS analysis was performed with an EASY-nLC 1000 LCsystem (ThermoFisher Scientific) interfaced via nanoSpray Flex ionsource to an Orbitrap Fusion MS (ThermoFisher Scientific). Briefly, thenLC was operated in a single analytical column set up using PicoFritEmitters (New Objectives, 75 μm inner diameter) custom packed withReprosil-Pure-AQ C18 phase (Dr. Maisch, 1.9-μm particle size, 19-21 cmcolumn length). Each sample was injected onto the column and eluted in agradient from 2 to 25% B in 45 min at 200 nL/min (Solvent A, 100% H₂O;Solvent B, 100% acetonitrile; both containing 0.1% (v/v) formic acid). Aprecursor MS1 scan (m/z 350-2,000) of intact peptides was acquired inthe Orbitrap Fusion at the nominal resolution setting of 120,000,followed by Orbitrap HCD-MS2 and at the nominal resolution setting of60,000 of the five most abundant multiply charged precursors in the MS1spectrum; a minimum MS1 signal threshold of 50,000 was used fortriggering data-dependent fragmentation events. Targeted MS/MS analysiswas performed by setting up a targeted MS^(n) (tMS^(n)) Scan Propertiespane.

Glycopeptide compositional analysis:

Analysis was performed from m/z features using in-house writtenSysBioWare software (Vakhrushev 2009). For m/z feature recognition fromfull MS scans LFQ Profiler Node of the Proteome discoverer 2.1(ThermoFisher Scientific) was used. A list of precursor ions (m/z,charge and retention time) was imported as ASCII data into SysBioWareand compositional assignment within 4 ppm mass tolerance was performed.The main building blocks used for the compositional analysis were:NeuAc, Hex, HexNAc, dHex and phosphate. The most prominent peptidescorresponding to each potential glycosites were added as an additionalbuilding block for compositional assignment. The most prominent peptidesequence related to each N-glycosite was determined experimentally bycomparing the yield of deamidated peptides before and after PNGase Ftreatment. A list of potential glycopeptides and glycoforms for eachglycosite was generated and the top 10 of the most abundant candidatesfor each glycosite were selected for targeted MS/MS analysis to confirmthe proposed structure. Each targeted MS/MS spectrum was subjected tomanual interpretation. Same N-glycan composition may represent isobaricstructures, so the listed glycan structure were assisted by and inagreement with literature data, predicted enzyme functions of thetargeted genes together with useful information in MS/MS fragments.

Result of site specific glycoanalysis of GLA produced in wt CHO cellsand the commercial Fabrazyme product is included in FIG. 2 , showingthat GLA from wt CHO cells was site-specifically glycosylated withmainly complex structures capped with SA at N-glycan site N108 and withhigh-mannose-type M6P glycans at sites N161 and N184

Examnle 2—Cell Based Glcoengineering of GLA Enzyme in CHO Cells

For producing improved enzyme the present inventors employednuclease-mediated knock-out or knock-in in CHO cells to engineer theglycosylation capacity, enabling production of lysosomal enzymes withimproved circulation time and/or improved targeting to affected organs.The genes targeted for modifying the glycans of the GLA enzyme arelisted in Table 1 and their role in the glycosylation process isincluded in FIG. 1 .

TABLE 1 List of gene modifications introduced into mammalian cells toimprove GLA enzyme. For knock-outs the gRNA's used for CRISPR/Cas9 engineering ofcells are included. Modifications may be introduced as single events or combinedwith other editing (stacked) Knock- out or SEQ Gene Gene Cell typeknock- ID name origin targeted in gRNA sequence NO Gnptg CHO CHO Knock-GCGATGGCGGTGCGGGTGGC 1 out Gnptab CHO CHO Knock- GTCACATTCATCGCATCGAG 2out St3gal4 CHO CHO Knock- GGTCGAAGTGGGCCGACTCA 3 out St3gal6 CHO CHOKnock- GGAGTTGTGATCATTGTGAG 4 out Mgat4B CHO CHO Knock-GAGAGGCAGGCGCTGCGGGA 5 out Mgat5 CHO CHO Knock- GACAATCTCGTCAATGGCAC 6out GNPTAB Human Human Knock- TAAACAACGTCAATCGGCAT 7 out GNPTG HumanHuman Knock- GCTGCTCACCCAAACGCGTT 8 out ST6GAL1 Human CHO + human Knock-na in ST3GAL4 Human CHO + human Knock- na in ST3GAL6 Human CHO + humanKnock- na in MGAT4A Human CHO + human Knock- na in MGAT4B HumanCHO + human Knock- na in MGAT5 Human CHO + human Knock- na inCRISPR/Cas9 Targeted Knock-Out in CHO Cells.

Gene targeting was performed in CHO clones stably expressing GLA(Example 1). Cells were seeded at 0.5×10⁶ cells/mL in T25 flask (NUNC,Denmark) one day prior to transfection, and 2×10⁶ cells and 1 μg each ofendotoxin free plasmid DNA of Cas9-GFP fusion and gRNA in the plasmidU6GRNA (Addgene Plasmid #68370) were used for electroporation usingAmaxa nucleofector as described in Example 1. 48 hours aftertransfection the 10-15% highest labeled (GFP) pool of cells was enrichedby FACS, and after 1 week in culture cells were single cell sorted into96-wells by FACS. KO clones were identified by Indel Detection byAmplicon Analysis (IDAA) as described in Lonowski 2017, as well as whenpossible by immunocytology with appropriate lectins or monoclonalantibodies. Selected clones were further verified by Sanger sequencing.The strategy enabled fast screening and selection of KO clones withframeshift mutations, and on average we selected 2-5 clones from eachtargeting event.

ZFNs/CRISPR-Mediated Knock-In in CHO Cells.

Site-specific CHO Safe-Harbor locus KI was based on ObLiGaRe strategyand performed with 2 μg of each ZFN (Merck/formerly known asSigma-Aldrich) tagged with GFP/Crimson as previously described (Yang2015), and 5 μg donor plasmid with full coding human genes (ST3GAL4,ST3GAL, ST6GAL1, MGAT4A, MGAT4B, MGAT5, GNPIAB, or GNPTG). In brief, theEPB69 donor plasmid contained inverted CHO Safe-Harbor locus ZFN bindingsites flanking the CMV promoter-ORF-BGH polyA terminator. Mono-allelictargeted KI clones with one intact allele were selected by IDAA analysis(Lonowski 2017). To stack a second gene into a Safe-Harbor locus, wefirst designed gRNA for the CHO Safe-Harbor locus flanking the ZFNbinding site, followed by transfection with 1 μg of a donor PCR productof gene to be inserted with 1 μg Cas9-GFP and 1 μg gRNA. In brief, thedonor PCR product was generated by using EPB69 donor plasmid as templatewhich contained the CMV promoter-ORF-BGH polyA terminator. KI cloneswere screened by PCR with primers specific for the junction area betweenthe donor plasmid and the Safe-Harbor locus. A primer set flanking thetargeted knock-in locus was used to characterize the allelic insertionstatus, and when possible, KI clones were also screened byimmunocytology with lectins and monoclonal antibodies.

Overall the gene targeting experiments did not affect viability, growth,or productivity substantially in the engineered cell clones, nor was GLAenzyme activity influenced by the modification of glycans.

Site specific glycoanalysis of GLA variants and Fabrazyme control isshown in FIG. 2 , demonstrating very similar glycosylation of Fabrazymeand GLA produced in wt-CHO cells, both in accordance with publication byLee 2003. The GLA-loM6P has lost M6P on site 2 (N161) but retains someM6P on third site, whereas the GLA-hiM6P has M6P on all three sites andno sialic acids. The GLA-hybM6P show hybrid glycan structure where sites1 and 3 have both M6P and SA, whereas site 2 only presents M6Pstructures. The two sialylated forms GLA-bi23SA and GLA-26SA both havelost M6P and all sites are dominated by biantennary structures with eachantenna capped with 1-2 sialic acids. The types of sialic acids arehomogeneous alpha2,3 or alpha2,6 respectively. FIG. 2 also depicts thevarious designs and glycostructures included in Table 2.

TABLE 2 The following five GLA glycovariants were selected and producedat larger scale for Fabry mouse studies: Cell Cell engineeringengineering GLA variant Design/Glycostructure Knock-out Knock-inGLA-bi23SA Biantennary, high SA, Gnptab, Gnptg ST3GAL4 2,3 linkageMgat4b/5 GLA-26SA Biantennary, high SA, Gnptab/g ST6GAL1 2,6 linkageSt3gal4/6 GLA-hiM6P High Man6P Alg3 GNPTAB GLA-loM6P Low Man6P Alg9 noGLA-hybM6P Hybrid with Man6P no GNPTAB GNPTG

Example 3—Role of Glycans on GLA Enzyme for PK and Biodistribution inMouse Model of Fabry Disease

For characterizing pharmacodynamics and potential therapeutic effects ofglycoengineered GLA a mouse model of Fabry disease was used forfollowing experiments. Specific enzyme activity for GLA produced in wtCHO cells and all GLA glycovariants was compared to Fabrazyme and nodifferences were found.

Experiment 1—Test PK and Biodistribution of GLA in Fabry Mouse Model:

Fabrazyme or CHO-GLA (GLA produced in CHO cells, Example 1) was injectedinto 3,5 month old male Fabry mice via tail vein at a dose of 1 mg/kgbody weight (n=4 mice per enzyme). Blood samples were collected by tailbleed at 5, 20, 40, 60 and 120 min after injection. Enzyme activity inplasma was measured, and the plasma clearance rate was determined. Fourhours after injection mice were sacrificed and liver, spleen, heart andkidney were harvested. Biodistribution was determined by measuring GLAactivity in the tissue lysates.

For pharmacokinetics the blood clearance of GLA produced in wt cells andFabrazyme was very similar (FIG. 3 ) and half-lives were 12.0 f 0.3 and11.9±2.3 minutes respectively. Overall the biodistribution among the 4major organs was similar albeit the GLA produced in our CHO cell linehad slightly lower activity in kidney (FIG. 4 ).

Experiment 2—Test PK and Biodistribution of Five Glycovariant Forms ofGLA in Fabry Mice:

The following enzyme variants were tested; GLA-bi23SA, GLA-26SA,GLA-HiM6P, GLA-LoM6P, GLA-HybM6P (see Table 2) and Fabrazyme wasincluded as control. Enzyme preparations (1 mg/kg) were iv injected into2 months old male Fabry mice via tail-vein (n=4 per group). At timepoints (5 min, 20 min, 40 min, 1 h, 2 h, 4 h and 24 h post-injection),small amount of blood samples were collected from tail vein. Plasma wasseparated by centrifugation, and was used for enzyme assay (seeExperimentl).

After 24 hours mice were sacrificed and heart, kidney, liver and spleenwere dissected. GLA activity was measured using standard 4MU method; thedata were expressed as nmol/hr/mg total protein.

The pharmacokinetics of four of the variants tested were similar toFabrazyme, however, the GLA-bi23SA variant showed a clearly prolongedblood clearance profile (FIG. 5 ) and an increased half-life by threefold (27.5±0.8 min versus 9.8±0.3 min for Fabrazyme) was observed. Thissuggests different uptake or clearance mechanisms for the GLA-bi23SAglycovariant with high SA of alpha2,3 type. The GLA-26SA variant, whichalso has high SA, but homogeneous alpha2,6 type, had same kinetics asFabrazyme and the other glycovariants and accordingly the alpha2,3linkage of the sialic acids is causal for increasing the plasmahalf-life.

FIG. 6 shows the distribution of GLA variants into heart, kidney, liverand spleen and based on this the relative distribution of GLA variantsinto the difficult-to-reach organs heart and kidney was calculated andis shown in FIG. 7 . The figures show that altering content of Man6P ofGLA (GLA-Lo/Hi/HybM6P) produce differences in organ distributions.Surprisingly, GLA with alpha2,3 SA and no Man6P (GLA-Bi23SA) showedclear uptake in organs with improved biodistribution characterized bylower levels in liver and spleen (FIGS. 6B and 6D) and significantlyhigher levels in heart compared to Fabrazyme (FIGS. 6A and 7 ). Thisdemonstrates for the first time that a lysosomal enzyme without M6Pand/or exposed Man are taken up by cells and distributed better tohard-to-reach organs compared to current lysosomal enzymes with Man6Pcontent. Moreover, surprisingly, GLA with alpha2,6 SA and no Man6P(GLA-26SA) showed clear uptake in organs with improved biodistributioncharacterized by higher levels in liver and significantly lower levelsin spleen and kidney compared to Fabrazyme. This demonstrates for thefirst time that a therapeutic glycoprotein with alpha2,6 SA on N-glycansselectively is distributed to the liver. FIG. 7 illustrates thesignificant changes in relative distribution of GLA variants tohard-to-reach organs showing highly improved biodistribution ofGLA-Bi23SA.

Example 4—Substrate Reduction in Fabry Mice

Gb3 is substrate for the GLA enzyme and accumulation of Gb3 in varioustissues is a characteristic feature of the Fabry disease. To test theefficacy of the GLA-Bi23SA for reducing Gb3 levels in selected organs inthe Fabry mouse the following experiment was performed.

GLA-Bi23SA or Fabrazyme enzyme preparations (1 mg/kg) or vehicle alone(saline) were injected into 6 months old female Fabry mice via tail-vein(n=5 per group). Age- and sex-matched untreated WT mice were used as WTcontrols (n=5). At 2 weeks after injection, heart, kidney and liver weredissected, and Gb3 levels were measured using mass-spectrometry. Thedata are expressed as ng/mg total protein (FIG. 8 ). In agreement withthe surprising biodistribution results the GLA-Bi23SA variant showedequivalent or better reduction in Gb3 content not only in heart, butalso in liver and kidney compared to Fabrazyme two weeks after thesingle dose of 1 mg/kg enzymes. Importantly, the lower levels ofGLA-Bi23SA activity distributed to liver and kidney did not adverselyaffect the decrease in Gb3 substrate in these organs compared toFabrazyme. The higher levels of GLA-Bi23SA activity distributed to theheart did not produce a significant lowering of Gb3 substrate comparedto Fabrazyme, although clearly a tendency towards lower detectablelevels were found (FIG. 8 ). These results clearly suggest that lowerdoses of GLA-Bi23SA and repeat infusions of GLA-Bi23SA will be superiorto Fabrazyme.

Example 5—Cellular Localization of Modified GLA (IHC)

For immunohistochemical analysis (IHC) enzyme preparations were injectedinto ˜3.5 month old male Fabry mice via tail-vein at a dose of 2 mg/kgbody weight. Heart, kidney and liver were harvested 24 h after enzymeinfusion. Untreated Fabry mouse tissues were used as negative controls.Tissues were fixed in formalin, embedded in paraffin, and 5-micronsections were made. IHC was performed by Histopathology and TissueShared Resource in Georgetown University (Washington, D.C.). In brief,after heat-induced epitope retrieval in citrate buffer, sections weretreated with 3% hydrogen peroxide and 10% normal goat serum, and wereincubated with rabbit polyclonal antibody to human α-gal A (Shire).After incubation with HRP-labeled secondary antibody, signals weredetected by DAB chromogen, and the sections were counterstained withhematoxylin. Signal specificity was verified with control staining, inwhich the primary antibody incubation was omitted.

Cellular localization of Fabrazyme and the glycovariants in the heart,kidney and liver was assessed by immunohistochemistry (IHC) (FIG. 9 ).The localization pattern of Fabrazyme in these organs was consistentwith that of agalsidase alfa reported in previous studies by Shen (2016)and Damme (2015). In the heart, Fabrazyme and all five glycovariantswere detected in vascular and/or perivascular cells, but not incardiomyocytes (FIG. 9C). There were no clear differences between thetested variants. In the kidney, Fabrazyme and GLA-LoM6P, GLA-HiM6P,GLA-HybM6P, and GLA-Bi23SA were predominantly detected in tubularepithelial cells (FIG. 9B). However, GLA-26SA had decreased number andintensity of positive signals in tubules compared to the other variantstested. In the liver, Fabrazyme, GLA-LoM6P, GLA-HiM6P, and GLA-HybM6Pwere detected in hepatocytes, putative Kupffer cells and endothelialcells of sinusoidal capillaries (FIG. 9A). GLA-Bi23SA was also detectedin these cell types; however, the number of positive signals inhepatocytes was decreased compared to Fabrazyme. Distribution ofGLA-26SA in the liver was remarkably different from the other variantsand Fabrazyme; GLA-26SA was detected almost exclusively in hepatocytes,and the number of positive signals in hepatocytes was clearly increasedcompared to Fabrazyme. This shows that the alphα2-6 SA glycoform can beused for selective targeting to hepatocytes.

In the heart all GLA variants produced distinct positive signals invascular and/or perivascular cells, but not in cardiomyocytes (FIG. 9C).All staining signals were with a granular cytoplasmic pattern indicatingcorrect localization to the lysosome.

Example 6—Production and Analysis of Glycoengineered GBA

Glucocerebrosidase (GBA) is a lysosomal β-glucosidase that degradesglucosylceramide. Its deficiency results in Gaucher disease (GD). Themature GBA enzyme is a 495 amino acids glycoprotein with four N-glycansites. Enzyme replacement therapy of macrophage-targeted recombinanthuman GBA markedly improves visceral symptomatology in GD patients, butthe inability of the infused enzyme to pass the blood-brain barrierprohibits the prevention and correction of neurological manifestations(Desnick 2012).

For producing recombinant human glucocerebrosidase A (GBA) we firstgenerated a stable high expressing CHO cell line. Full length cDNA ofhuman GBA1 with His tag was purchased from Sino Biological Inc. andsubcloned into modified pCGS3 (Merck/Sigma-Aldrich) for glutamineselection in CHOZN GS−/− cells. Subsequent selection and isolation of aclonal cell line producing GBA was performed as described in Example 1.

For optimization the GBA glycodesigns shown in Table 3 were planned andcell engineering was done according to protocol described in example 2.Cell clones were analyzed using GBA ELISA assay.

TABLE 3 The following GBA glycovariants were pursued: Cell Cellengineering engineering GBA variant Design/Glycostructure Knock-outKnock-in GBA-23SA No M6P, 2,3 SA Gnptab no linkage GBA-hi23SA No M6P,high SA, 2,3 Gnptab ST3GAL4 linkage GBA-HiMan No M6P, high man Gnptab,Mgat1 no GBA-Hybrid No M6P, hybrid with 3 Gnptab, no (3 mannoses) man onalpha 1,6 Man2a1/2 branch GBA-Hybrid No M6P, hybrid with 1 Gnptab, Mgat2no (1 mannose) man on alpha 1,6 branch

Example 7—Glycoengineering of GLA in Human Cells

For glycoengineering in human cells (like HEK293) the knock-outs weregenerated using gRNA addressing human genes. For all humanglycosyltransferase genes optimized gRNA's were described in Narimatsu2018 and for human GNPTAB and GNPTG optimized gRNA sequences areincluded in Table 1. Human cells predominantly produce alpha2,6 sialicacid capping on the N-glycans and therefore for obtaining homogeneousalpha2,3 SA capping knock-out of the alpha2,6 capping enzyme ST6GAL1 isneeded.

The following engineering is performed:

-   -   1) HEK293 with knock-out of GNPTAB    -   2) HEK293 cell with knock-out of GNPTAB/ST6GAL1    -   3) HEK293 cell with knock-out of GNPTAB/ST6GAL1 and knock-in of        ST3GAL4    -   4) HEK293 with knock-out of GNPTG/GNPTAB/ST6GAL1 and knock-in of        ST3GAL4    -   5) HEK293 with knock-out of GNPTG/GNPTAB/ST6GAL1/MGAT4B/MGAT5        and knock-in of ST3GAL4

For transient expression in HEK293 cells an expression constructscontaining the entire coding sequence of human GLA was cloned into BamH1site of the pTT5 expression vector (Durocher 2002). Engineered HEK293-6Ecells were cultured in DMEM/high glucose medium supplemented with 10%FBS and 1% Glutamax. 60% confluent cells were seeded in T75 flasks theday prior to transfection. Plasmid was transfected into cells using PEI,by mixing 30ul PEI (0.1% linear 25k Polyethylenimin in 150 mM NaCl, pH7.0) with 10ug-GLA.pTT5 expression plasmid in 2 ml Opti-MEM Medium. Oneday after transfection, culture medium was changed to F17 Mediumsupplemented with 2% Glutamax and 1% TN1 (Tryptone Ni). Culturesupernatant was collected after incubating the cells at 37 C for another2 to 3 days in F17 medium. Secreted GLA was purified from culturesupernatant by ion exchange on a DEAE column. The culture supernatantwas centrifuged at 3,000 g for 20 min and then further filtered forclarification through a 0.45 m filter. After dilution with 3 volumes of25 mM MES (pH 6.0) the resulting solution was loaded onto a DEAEsepharose fast-flow column pre-equilibrated with the same buffer.Elution was carried out by applying 0.2 M sodium chloride in 25 mM MES(pH 6.0) and the fractions containing the recombinant GLA weredetermined by enzyme activity assay and collected. Purity and roughtiter of GLA was evaluated by Coomassie staining of SDS-PAGE gels. Sitespecific N-glycan profiling was done according to procedure described inExample 1.

Site specific glycoprofiling of GBA produced in wt HEK293 cells orHEK293 cells with GNPTAB knock-out is shown in FIG. 10 . The capping ofwt glycans produced in HEK293 are similar to CHO (FIG. 2 ) albeit theCHO cells produced more tri-tetra antennae where HEK293 mostly producebiantennary forms. The knock-out of GNPTAB abolishes the Man6P cappingon sites 2 and 3 which were major forms in wt GBA.

Example 8—GLA with Increased Sialylation (Tri/Tetra Antennary)

To increase sialic acid content of a lysosomal enzyme the total numberof antennae on the glycans may be increased. For increasing number ofantennae and obtain higher alpha2,3SA content, the inventors generateCHO cell lines with various combinations of MGAT4A/MGAT4B/MGAT5/ST3GAL4knock-in combined with knock-out of Gnptab and/or gnptg. Theseengineered cell lines are used to produce novel GLA glycovariants, andvariants with most promising glycoforms are analyzed in PK/PD mousestudies. For example, as is discussed further in Example 16 and shown inFIG. 27A, GLA sialic acid content may be optimized by producing theenzyme in CHO cells containing knock-outs of Gnptab and MGAT4B/5 andfurther containing knock-in of ST3GAL4 and MGAT4A/5. Such aglycostructure-optimized GLA enzyme comprises no Man6P and hashigh-antennary glycans with high 2,3 sialic acid content, and thesechanges result in higher enzyme activity in plasma compared to GLAproduced in unmodified wild-type CHO cells (FIG. 27A). Other GLAglycostructures are similarly produced as disclosed herein, to modulatePK/PD properties.

Example 9—Cell Based Glycoengineering of Other Lysosomal Enzymes

Optimized glycans may be displayed on any lysosomal enzyme including,e.g., human iduronate 2-sulfatase (IDS), human arylsulfatase B(N-acetylgalactosamine-4-sulfatase) (ARSB), human lysosomalα-glucosidase (GAA), human alpha-galactosidase (GLA), humanbeta-glucuronidase (GUSB), human alpha-L-iduronidase (IDUA), humaniduronate 2-sulfatase (IDS), human beta-hexosaminidase alpha (HEXA),human beta-hexosaminidase beta (HEXB), human lysosomal α-mannosidase(mannosidase alpha class 2B member 1) (MAN2B1), human glucosylceramidase(GBA), human lysosomal acid lipase/cholesteryl ester hydrolase (lipaseA, lysosomal acid type)(LIPA), human aspartylglucosaminidase(N(4)-(beta-N-acetylglucosaminyl)-L-asparaginase) (AGA), and humangalactosylceramidase (GALC), by producing such enzymes in the modifiedcells disclosed herein, e.g., CHO cells or HEK cells containing knock-inand/or knock-out of glycosyltransferase genes.

For example, as is discussed further in Example 16 and shown in FIGS.27B-D, the lysosomal enzymes GUSB, AGA and Laman were optimized byproducing these enzyme in CHO cells containing variousglycosyltransferase gene knock-outs and, optionally knock-in to produceoptimized enzymes with no Man6P and, optionally, High 2,3, SA, resultingin higher enzyme activity in plasma compared to enzyme produced inunmodified wild-type CHO cells. Thus, these data demonstrate that theoptimized glycodesign methods disclosed herein are broadly applicable toany lysosomal enzymes. Such modified enzymes may be useful in treatinglysosomal storage diseases such as, e.g., any one or more of those shownin Table 4.

TABLE 4 Lysomal Storage Diseases and Contributing Defective EnzymesLYSOSOMAL STORAGE DISEASE DEFECTIVE ENZYME AspartylglucoaminouriaAspartylglucoaminidase (AGA) (AGU) Fabry Alpha-Galactosidase A (GLA)Farber Acid ceramidase Fucosidosis Acid alpha-L-fucosidaseGalactosidosialidosis Protective protein/Cathepsin A Gaucher types 1, 2,and 3 Acid beta-glucosidase, or glucocerebrosidase (GBA) G-M1gangliosidosis Acid beta-galactosidase Hunter Iduronate-2-sulfatase(IDS) Hurler-Scheie Alpha-L-Iduronidase (IDUA) KrabbeGalactocerebrosidase/galactosylceramidase (GALC) Alpha-Mannosidosis Acidalpha-mannosidase (Laman) Beta-Mannosidosis Acid beta-mannosidaseMaroteaux-Lamy Arylsulfatase B Metachromatic Arylsulfatase A Morquio BAcid beta-galactosidase Mucolipidosis II/IIIN-Acetylglucosamine-1-phosphotransferase Pompe Lysosomalalpha-glucosidase (GAA)

Example 10—Identifying Optimal Glycovariant for a Lysosomal Enzyme

A systematic approach for determining which glycomodification thatinfluence and improve activity of an lysosomal enzyme, may comprise thefollowing:

-   -   1) Generating a multiplicity of mammalian cells with        modification of genes resulting in modified N-glycans (high/low)        with respect to the following parameters: M6P content, Sialic        Acid content, Ratio between α2,3/α2,6 Sialic acids, and exposed        Mannose content.    -   2) Express the enzyme in the glycoengineered cell lines and        produce a multiplicity of glycovariants of the enzyme    -   3) Screen the multiplicity of enzyme glycovariants for optimized        drug effect in relevant in-vitro assay and/or animal model    -   4) Identify which glycovariants (and glycodesigns) that have        improved drug function

Additional round(s) of glycoengineering and screening (steps 1-4) may beapplied to secure optimal glycovariant candidate.

The optimization aims at identifying a glycovariant of the enzyme whichultimately give improved clinical performance with respect to one ormore parameters including efficacy, dosing, potency, purity, lessside-effects and better safety. The assays used for screening willmonitor biomarkers/reporters for one or more of these parameters.

For an enzyme glycovariant with improved drug function a production cellline may be developed by transferring the glycodesign to any mammaliancell based production platform.

The examples demonstrate application of a novel cell basedglycoengineering platform to generate lysosomal enzymes with improvedbiodistribution. The cell based production of the final modified enzymeenable easy transfer into industrial manufacturing facilities and rapidand cost-effective development of novel improved ERT's for lysosomalstorage diseases

Example 11—Stabilizing the Optimized GLA Variant with1-Deoxygalactonojirimycin (DGJ)

For stabilizing the glycooptimized GLA-bi23SA it was mixed and/orcoformulated with 1-deoxygalactonojirimycin (DGJ).

Physical stability of modified GLA is established by thermal stabilityassay e.g. using method described in Benjamin 2012. Briefly GLA wascombined with fluorescent SYPRO Orange and various concentrations of DGJand fluorescence was monitored over time.

In vitro plasma stability of the modified GLA was determined by mixingGLA (1 mg/ml) and DGJ (1/3/10 mg/ml) and mixing with plasma (mouse orhuman). GLA activity was measured at various time points and in-vitroplasma half-life was established.

In-vivo stability and pharmacokinetics of modified GLA with varyingconcentrations of DGJ was established by coformulation andadministrating modified GLA (0.1/0.2/0.5 or 1.0 mg/kg) with DGJ(0.1/0.3/0.5/1/3/10 mg/kg) to Fabry mice. Pharmacokinetics wasestablished as described in Example 3.

Example 12—the Glycosylation Design Space for Recombinant LysosomalReplacement Enzymes in CHO Cells

Lysosomal replacement enzymes are essential therapeutic options for rarecongenital lysosomal enzyme deficiencies, but enzymes in clinical useare only partially effective due to short circulatory half-life andinefficient biodistribution. Replacement enzymes are primarily taken upby cell surface receptors recognizing glycans, and the structures ofglycans influence uptake, biodistribution, and circulation time. It hasnot been possible to design and systematically study effects ofdifferent glycan features in the past, but here we present acomprehensive gene engineering screen in Chinese hamster ovary (CHO)cells that for the first time enables production of lysosomal enzymeswith N-glycans custom designed to affect key glycan features guidingcellular uptake and circulation. We demonstrate distinct predicted organdistribution and circulation time of different glycoforms ofα-galactosidase A in a Fabry disease mouse model, and find that an α2-3sialylated glycoform designed to eliminate uptake by the mannose6-phosphate and mannose receptors exhibits improved targeting tohard-to-reach organs such as heart, and longer circulation time. Thismay suggest a paradigm shift in design of some replacement enzymes, andthe developed design matrix and engineered CHO cell lines now enablessystematic studies towards improving enzyme replacement therapeutics.

Lysosomal storage diseases (LSDs) are characterized by the progressiveaccumulation of undegraded metabolites that lead to lysosomal andcellular dysfunction. At present ˜70 LSDs are known and most are causedby inherited gene mutations that impair targeting or function oflysosomal enzymes. During the past three decades, a variety oftherapeutic approaches have been developed for LSDs with intravenousenzyme replacement therapies (ERTs) being the most prevalent, andcurrently replacement enzymes for Fabry disease, Gaucher disease type I,mucopolysaccharidoses (MPS I, II, IVA, VI and VII5), Neuronal CeroidLipofuscinosis (CLN2)6, Wolman disease, and Pompe disease are in use,and more are in clinical trials. Despite more than 25 years of clinicalexperience, ERTs for LSDs still face major challenges, and the mostimportant may be the delivery of the infused recombinant enzymes tohard-to-reach organs such as bone, cartilage, kidney, heart, and brain.

Lysosomal enzymes are glycoproteins with N-glycans and the cellularuptake of replacement enzymes is thought to primarily rely oncell-surface receptors recognizing N-glycan features. A number ofglycan-binding receptors are found on different cell types including themannose 6-phosphate (M6P) receptors (MPRs), Ashwell-Morell receptor(AMR) or asialoglycoprotein receptor (ASGPR), and mannose receptor (MR),and these participate in cellular uptake and lysosomal delivery oftherapeutic glycoproteins. The MPRs specifically recognize terminal M6Pattached to high-mannose and hybrid-type N-glycans, and they direct bothintracellular delivery of endogenously produced lysosomal enzymes aswell as the uptake and delivery of exogenous M6P-containingglycoproteins from circulation and extracellular space. The AMRexpressed primarily on liver hepatocytes recognizes glycoproteins withuncapped terminal galactose (Gal) or N-Acetyl-galactosamine (GalNAc)residues, usually as a result of insufficient addition of sialic acidsor loss due to sialidase activities, and mediates rapid clearance fromthe circulation. The MR expressed primarily on mononuclear macrophagesbinds mainly exposed mannose (Man), N-Acetyl-glucosamine (GlcNAc) andfucose (Fuc) residues on N-glycans, and directs uptake of glycoproteinsand targeting to endosomes and lysosomes. Tissue distribution andcirculation time of infused replacement enzymes are at least partlydependent on the expression of these receptors. A number of otherglycan-binding proteins and receptors including Siglecs and Galectinscould potentially bind therapeutic N-glycoproteins, andglycan-independent uptake of lysosomal enzymes by e.g. the low-densitylipoprotein receptor proteins (LRPs). The glycosylation states of mostreplacement enzymes are critical for the pharmacokinetic properties andtherapeutic effect, with the key determining factors believed to be abalance between N-glycan features that include the degree of M6P-taggingand exposure of terminal Man, Gal, and/or GlcNAc residues in a complexinterplay yet unexplored. Most currently approved ERTs have highlyheterogeneous N-glycan structures, often with low M6P stoichiometry, asdictated by the inherent glycosylation capacity of CHO cells. However,there have been limited options for custom design of the glycosylationcapacity of CHO cells and thus for testing specific ERTs with differentN-glycan features to explore potential improved therapeutic performance.

Different strategies have been undertaken to explore glycoengineering asa means to improve delivery of ERTs, including use of exoglycosidasesfor postproduction enzyme modification, as well as the use of engineeredyeast and plant production platforms. Pioneering work originallydemonstrated how glycosidase trimming of N-glycans on the lysosomalenzyme β-glucoscerebrosidase (GBA) resulted in efficient targeting ofmacrophages through the MR and provided a successful therapy fornon-neuropathic Gaucher disease. The first recombinant GBA replacementenzyme was produced in CHO cells with Man terminated (high-Man)N-glycansby postproduction exoglycosidase treatment, and other two producedeither in human cells by use of an inhibitor of mannosidase I(kifunensine) or in carrot cells. Most strategies for glycoengineeringof lysosomal enzymes have sought to improve targeting by MPRs and MRs byincreasing the content of M6P or exposed Man residues. However, theseglycoforms will cause rapid and efficient uptake by especially the liverand spleen, while targeting to other organs may be limited. Earlystudies have demonstrated that increased content of sialic acid (SA) onlysosomal enzymes isolated from plasma improves their circulation timesimilar to other types of therapeutic glycoproteins. Further studies ofsuch glycoforms have been hampered by lack of suitable methods toproduce these recombinantly. A different strategy to eliminatelectin-mediated interactions by MPRs, MR and other lectins including theAMR employing oxidative degradation and reduction of recombinant enzymeshas demonstrated therapeutic efficacy with extended circulation time andwider biodistribution in some cases. However, the procedure partlyinactivates the enzyme and may not be easy to control for clinicalproduction.

Most enzymes used for ERTs are produced in CHO cells, and with theadvent of efficient precise gene editing tools it is now possible tointroduce extensive engineering designs to optimize the glycosylationcapacity of CHO cells. Here, we present a first comprehensive screen ofengineering options for lysosomal enzymes in CHO cells, and we provide apanel of glycoengineered CHO cell lines with different capacities forproducing lysosomal enzymes furnished with all the key glycan featuresknown to affect cellular uptake and circulation time. The extensiveengineering performed provides a genetic design matrix that makes itpossible to produce and investigate optimal glycoform(s) withhigh/low/no M6P-tagging, varying exposure of Man, Gal, and GlcNAcresidues, and different capping by SA for any lysosomal replacementenzyme without the need for using postproduction enzyme modifications oralternative yeast and plant expression systems. Using theα-galactosidase A (GLA) as a representative of lysosomal enzymereplacement enzyme therapeutics in a mouse model of Fabry disease, wedemonstrate how distinct glycoforms of GLA are differentially targetedto liver, spleen, kidney and the heart. We tested the performance ofglycoforms without M6P and exposed Man residues to explore glycoformswithout ligands for the major MPRs and MR receptors, and present thefirst evidence that glycoforms capped with α2-3 linked sialic acids(α2-3SA), but surprisingly not α2-6SA, exhibit improved circulation andbiodistribution, and importantly with higher uptake in heart compared tothe current leading agalsidase beta (Fabrazyme) ERT. Thus, in contrastto the current dogma α2-3SA capped glycoforms of at least lysosomalenzymes may represent a new strategy to overcome the most criticalproblems of rapid clearance in liver and poor biodistribution found withcurrent ERTs.

Results

Exploring the Glycoengineering Design Space for Lysosomal EnzymesProduced in CHO Cells

We first established a stable wildtype (WT) CHO clone expressing highlevels of human GLA, which is one of the most widely used ERTs today.Next, we performed a clustered regularly interspaced short palindromicrepeat and CRISPR-associated protein 9 (CRISPR/Cas9) mediated geneknockout (KO) targeting screen in the GLA expressing CHO cell lineconsidering all glycosyltransferases and glycosylhydrolases with knownor putative functions in N-glycosylation and M6P processing pathways,including enzymes involved in assembly of the lipid-linkedoligosaccharide precursor (FIG. 11 ). We also targeted the MPRs andother receptors known to direct transport of lysosomal enzymes, as wellas a heterogeneous group of enzymes known to affect stability andprocessing of glycosyltransferases (FIG. 11 ). RNAseq expressionprofiling was used to identify relevant genes expressed in CHO cells(FIG. 16 ). We designed and tested 3-4 gRNAs per gene for all such geneswith a high throughput work-flow as reported previously (Narimatsu, Y.et al. A validated gRNA library for CRISPR/Cas9 targeting of the humanglycosyltransferase genome. Glycobiology 28, 295-305 (2018),incorporated herein by reference in its entirety). GFP-tagged Cas9nuclease was used to enrich for high Cas9 expression by FACS, and thecutting efficiency and indel profile of each gRNA characterized by IndelDetection by Amplicon Analysis (IDAA)(Yang, Z. et al. Fast and sensitivedetection of indels induced by precise gene targeting. Nucleic Acids Res43, e59 (2015), incorporated herein by reference in its entirety). Wedeveloped 43 validated gRNAs constructs (Table 5) and more than 200 CHOcell clones with different gene engineering (Tables 6 and 7). Ingeneral, the gene targeting did not affect viability, growth, orproductivity substantially in the mutant cell clones.

TABLE 5CRISPR gRNA design and list of PCR primers used for gRNA target sites.Gene gRNA #* Forward primer (5′-3′) #* Reverse primer (5′-3′) #* Acp2GCTCTGCGGCAGCGCTATAG 9 TCGTCTCTTCCCAGACAAGC 52 TAGGGTCTGTGAGCCATCCC 95Acp5 GGATGCACGGACGGTACTGC 10 GTGCAGTTTTCAGGGGCTTG 53CTCCCCAGAGTAAGGTCCCA 96 Alg1 TTCTGCAAGAGCTCATCTCG 11CCCCAGTACACAACTACCCC 54 AGTACATGCTGGCCTTGAACA 97 Alg2CTGTGACGTGAAGATATGGA 12 CTGCTGCTGGACAGTTCCAA 55 ATTGCAGAAGCTCGAGCGAA 98Alg3 GCTGCTGGGCTGCGGAAACG 13 TAGCTAGAAACCCTGGTGCC 56TAGTGAACTCACATGCCACCC 99 Alg5 GGACTCTAAGTTCACTTACG 14TAGTAGGAGAGAGCCGACCC 57 CTTGGGTTCCTCCAGCAAGT 100 Alg6TCTTAATAGGACTCACAGTG 15 AAGCAGATGCAGCCCACTCA 58 CAAGTGACGGACTTAGCAGGA101 Alg8 TCGGTGTACTTCAAAATCCG 16 GTGCAGTGGTCTAAGAACCCA 59TCAAGGCCTGGCAGCTTAC 102 Alg9 GAGCAGACATTTGAAAGCAG 17GCCCAAGACCATCGGTTAGAT 60 TGTCCGGATTTAGTCTTCGCT 103 Alg11ACTGGTGACATTAATGTCAG 18 TGAGTCCCTTTCTTTTTGTGCC 61 TCAGGAACACGCTGTGTCAG104 Alg12 AAATCACCAGGCAAGTCAGG 19 CAGTGTGACCTTAAGCAGGGT 62CAGGTCATGCGTAGCCTGTA 105 Alg13 GATCTTGTCATCAGCCACGC 20AGTTATGAACCACGGAGCCA 63 TTGGAAGCTTAGCCAACTGGT 106 Alg14CTGCGGCAGCTAGAATCAGG 21 TTTGACCGCCCAACTCATCA 64 AGCGCTCGTAAAGGTGCTAA 10784galt1 GGGCGGTCGTTATTCCCCCA 22 CCCAAACCTCACCTGGTTGAT 65GCTGGCTAACATCTTCGTTCC 108 84galt3 GCAGGACGGTACCGGCCCCC 23ATGCCATATGCAAGCTGCTG 66 GTGGGTCCTGTGTCGGTATC 109 Fam20cGGGAAGCCTGACCAGATCGA 24 ATAGGTCACCGACTCTCCCT 67 GCCAATAACATCTGCTTCTACGG110 Furin GACCAAGCGGGACGTGTATC 25 GCCCATCTCGGTCTCATTGC 68TGGGGAAGAAGACCAGAACCC 111 Fut8 GATCCGTCCACAACCTTGGC 26AGAGTCCATGGTGATCCTGC 69 TACTGTTTAAGGGGAGGGGGA 112 GanabGAAGGCTTCGATCCTCTAGC 27 GTCGTCTTGCCAACCCCAAA 70 CACACCCAGTCTCTTCCCAA 113Gnptab GTCACATTCATCGCATCGAG 28 ACCAACGGGCAGATTCCTTC 71CTAGGTGCCCACCCATCTTAG 114 Gnptg GCGATGGCGGTGCGGGTGGC 29CTTCCGGTTTTGAGCGCAG 72 CAGCCAAGGGCTTTCCTCG 115 Golph3GAAAGGCTCAGTGCAACACT 30 GCACAACTGACTCCAGGATG 73 GAGCTCTTCAGATGCCATAACC116 Golph3l TGACTTCAGTTCGACGGGTA 31 CTCTTTCCCATGTTCCTCCA 74TGTGTGTGTATAGGTCTTCTGTGG 117 Igf2r GACAAAAACCTGTCGATCAG 32GCTACACATGGGAGGCTGTT 75 CAAACCCAAAGCTGCGGAAA 118 Lrp2TCACACAAGGAATTCCAGTG 33 ATCAGTGCCCACTGCCTAAC 76 AAGGAACCCAGGTCAAGCAA 119M6Pr GCTATAGATTCAGAGTATGC 34 AAGGGAGGGGTGCAGTTTTT 77GACCAGCTGTGGAACTAGGC 120 Man1a1 GTAAATATACGCTTCGTCGG 35TGGGCAAGCACACAGGTTTA 78 TGACCACCGGAACACGAAAA 121 Man1a2GTCTGTGTTCGTCGGGTCCA 36 CCACAGGGCTACCTTGAGAC 79 GTCCTTCCAGGCTATGGCTC 122Mon1b1 GAGTACATACCACCTATCGG 37 AGCACCAGCACAAAGGGATT 80GCTTTCACCCTCTCATTACGC 123 Man1c1 GCCCCGGGCGAGGACGATCC 38TGGAGGTGATGGCCGAAAAC 81 CAGTGTGCCTGAAGGGTCTC 124 Man2a1GAGTGAAGCCTCGATCGGGT 39 TAATCACAGCTGCGAGGTGG 82 ACTGCTATGCACCCCCATTC 125Man2a2 GCCCAGAGAAAGCGTCGTCG 40 AGCGGCATATTCAGGGGAAC 83GGGACTGCATACATTGGCCT 126 Monea ATAGCCAAGAACTATCCACA 41CGCCCCCTTGGAAAACAAA 84 CGAACTAATTACCAACCAATTGAGG 127 Mgat1GAGGGGGTCGCAGGCACACG 42 GTGCTTTGGGGTGCTATCCT 85 TGTGACTGCACTGCCATAGG 128Mgat2 GCGACCGGTACCGCAGCGTT 43 GCGACAAAGGAAGAACGACG 86TAGGTCTCTGGGGCAGTCTC 129 Mgat4b GAGAGGCAGGCGCTGCGGGA 44TAGCCTGTGTGTGTCAACCC 87 TGGGGAAGGGACAGGTTAGA 130 Mgat5GACAATCTCGTCAATGGCAC 45 ACCTGCAGAGGTTTTCAGTTCT 88 GCCTTCACAACAATCATGCCA131 Mogs GGTGTCCCTGTTCTTCTACG 46 TTTAGCTCAGCCCACTCCAG 89CTCCCTACCCGTACCACTCT 132 Nagpa GGGCTGCAGAACGCGCAGTT 47AGAACGGTGGTTTCTTCCGC 90 GCGTTCAATGACACACGACT 133 Sort1TTAACAGCAGAGGTATCTGG 48 AGGACCATGCCCTGCTCTC 91 ATAGCCAGATGGGGACAGGTAG134 Sppl3 GAGGCTTGGCAGGCGGACAA 49 ATGTCACCGACAAACGGGAC 92CCACACACCAACTGATCCCC 135 St3gal4 GGTCGAAGTGGGCCGACTCA 50AAGAGCGTGTCTGGGTTGTT 93 GCAGGGTCCACTTCTGGATT 136 St3gal6GGAGTTGTGATCATTGTGAG 51 TCTTGGGTGCTTCTGAGTGTG 94 GGACACAGAAAATGGGATGTTG137 *In Table 5, # denotes the SEQ ID NO of the given gRNA or primersequence.

TABLE 6 Summary of CHO mutant clones stably expressing GLA and cell lineancestry. Project Parental CHO number Targeted genes line FAB399 KONagpa WT#H9* FAB400 KO Gnptab WT#H9 FAB441 KO Igf2r WT#H9 FAB442 KOMan1a1 WT#H9 FAB443 KO Man1a2 WT#H9 FAB444 KO Man1b1 WT#H9 FAB445 KOMan1c1 WT#H9 FAB446 KO Mogs WT#H9 FAB451 KO Acp2 WT#H9 FAB453 KO GnptgWT#H9 FAB454 KO Acp5 WT#H9 FAB462 KO Acp2/5 WT#H9 FAB478 KO Alg3 WT#H9FAB479 KO Alg6 WT#H9 FAB480 KO Alg9 WT#H9 FAB494 KO St3gal4/6 WT#H9FAB495 KO Mgat1 WT#H9 FAB496 KO Mgat2 WT#H9 FAB497 KO Mgat4b/5 WT#H9FAB499 KO Sppl3 WT#H9 FAB532 KI ST6GAL1/KO St3gal4/6 FAB494 B4 FAB534 KOSort1 WT#H9 FAB535 KO Lrp2 WT#H9 FAB540 KO Fut8 WT#H9 FAB546 KO Gnptab/gWT#H9 FAB555 KO Furin WT#H9 FAB560 KO Manea WT#H9 FAB567 KOMgat4b/5/Gnptab/g FAB546A2 FAB568 KO Mgat1/Gnptab/g FAB546A2 FAB570 KOMgat2/Gnptab/g FAB546A2 FAB571 KO B4galt1/3/Mgat4b/5/Gnptab/g FAB567H3FAB572 KO Ganab WT#H9 FAB583 KI ST3GAL4/KOMgat4b/5/Gnptab/g FAB567H3FAB584 KO Fut8/Mgat4b/5/Gnptab/g FAB567H3 FAB604 KO Fam20c WT#H9 FAB605KO Golph3 WT#H9 FAB606 KO Golph3l WT#H9 FAB611 KO Alg3/Mgat1 FAB495B10FAB662 KO Alg8 WT#H9 FAB664 KO Alg12 WT#H9 FAB667 KO Alg5 WT#H9 FAB677KI GNPTG WT#H9 FAB688 KO B4galt1/3 WT#H9 FAB695 KI GNPTAB WT#H9 FAB712KO Man2a1/2 WT#H9 FAB713 KO Man2a1/2/Gnptab FAB400C7 FAB725 KOMan1a1/1a2/1b1/1c1 FAB442G8 FAB791 KO M6pr WT#H9 FAB792 KI GNPTAB/KOAlg3 FAB695G2 FAB793 KI GNPTAB/KO Alg3 FAB695A8 FAB819 KI GNPTAB/GFAB695G2 FAB857 KI ST6GAL1/KO St3gal4/6/Gnptab FAB532D2 FAB870 KIST6GAL1/KO St3gal4/6/Gnptab/Fut8 FAB857D2 *Clone WT#H9 was used asparental clone for all gene engineering.

TABLE 7 Sequence analysis of CHO mutant clones stably expressing GLA.Targeted Clone genes InDel Alignment SEQ ID NO: FAB399C3 KO Nagpa WTGGGCTGCAGAACGCGCAGTTCGG 138 KO  +1 bp GGGCTGCAGAACGCGCAaGTT CGG 139FAB400C7 KO Gnptab WT GTCACATTCATCGCATCGAGGGG 140 KO  +1 bpGTCACATTCATCGCATCcGAG GGG 141 FAB441C2 KO Igf2r WTGACAAAAACCTGTCGATCAGTGG 142 KO  +1 bp GACAAAAACCTGTCGATcCAG TGG 143FAB442G8 KO Man1a1 WT GTAAATATACGCTTCGTCGGTGG 144 KO  -2 bpGTAAATATACGCTT

TCGG TGG 145 FAB443C5 KO Man1a2 WT GTCTGTGTTCGTCGGGTCCATGG 146 KO  +1 bpGTCTGTGTTCGTCGGGTtCCA TGG 147 FAB444E7 KO Man1b1 WTGAGTACATACCACCTATCGGGGG 148 KO  +1 bp GAGTACATACCACCTATtCGG GGG 149FAB445H5 KO Man1c1 WT GCCCCGGGCGAGGACGATCCCGG 150 KO  +1 bpGCCCCGGGCGAGGACGAaTCC CGG 151 FAB446A2 KO Mogs WTGGTGTCCCTGTTCTTCTACGTGG 152 KO  +1 bp GGTGTCCCTGTTCTTCTtACG TGG 153FAB451H7 KO Acp2 WT GCTCTGCGGCAGCGCTATAGTGG 154 KO  +1 bpGCTCTGCGGCAGCGCTAaTAG TGG 155 FAB453C1 KO Gnptg WTGCGATGGCGGTGCGGGTGGCCGG 156 KO −57 bp CAGTTC--

-- 157 TTCCT FAB454F2 KO Acp5 WT GGATGCACGGACGGTACTGCTGG 158 KO  +1 bpGGATGCACGGACGGTACcTGC TGG 159 FAB454F2 KO Acp5 WTGGATGCACGGACGGTACTGCTGG 160 KO  +1 bp GGATGCACGGACGGTACcTGC TGG 161FAB462C1 KO Acp2 WT GCTCTGCGGCAGCGCTATAGTGG 162 KO  -2 bpGCTCTGCGGCAGCGC

TAG TGG 163 KO Acp5 WT GGATGCACGGACGGTACTGCTGG 164 KO-alle1  -1 bpGGATGCACGGACGG ACTGC TGG 165 KO-alle2 −20 bp AGATGG--

-- 166 TGTCA FAB462B2 KO Acp2 WT GCTCTGCGGCAGCGCTATAGTGG 167 KO  +1 bpGCTCTGCGGCAGCGCTAtTAG TGG 168 KO Acp5 WT GGATGCACGGACGGTACTGCTGG 169 KO +1 bp GGATGCACGGACGGTACgTGC TGG 170 FAB478H2 KO Alg3 WTGCTGCTGGGCTGCGGAAACGCGG 171 KO  +1 bp GCTGCTGGGCTGCGGAAaACG CGG 172FAB478F5 KO Alg3 WT GCTGCTGGGCTGCGGAAACGCGG 173 KO  -1 bpGCTGCTGGGCTGCGGA

ACG CGG 174 FAB479A8 KO Alg6 WT TCTTAATAGGAC TCACAGTGCGG 175 KO  +1 bpTCTTAATAGGACTCACcAGTG CGG 176 FAB480C2 KO Alg9 WTGAGCAGACATTTGASAAGCAGTGG 177 KO  +1 bp GAGCAGACATTTGAAAGgCAG TGG 178FAB494B4 KO St3gal4 WT GGTCGAAGTGGGCCGACTCAGGG 179 KO  +1 bpGGTCGAAGTGGGCCGACcTCA GGG 180 KO St3gal6 WT GGAGTTGTGATCATTGTGAGCGG 181KO  +1 bp GGAGTTGTGATCATTGTtGAG CGG 182 FAB495B10 KO Mgat1 WTGAGGGGGTCGCAGGCACACGGGG 183 KO  +1 bp GAGGGGGTCGCAGGCACgACG GGG 184FAB496E4 KO Mgat2 WT GCGACCGGTACCGCAGCGTTAGG 185 KO  +1 bpGCGACCGGTACCGCAGCcGTT AGG 186 FAB497G4 KO Mgat4b WTGAGAGGCAGGCGCTGCGGGACGG 187 KO  +1 bp GAGAGGCAGGCGCTGCGgGGA CGG 188KO Mgat5 WT GACAATCTCGTCAATGGCACAGG 189 KO  -1 bp GACAATCTCGTCAATG

CAC AGG 190 FAB499B1 KO Sppl3 WT GAGGCTTGGCAGGCGGACAAAGG 191 KO  +1 bpGAGGCTTGGCAGGCGGAaCAA AGG 192 FAB532D2 KO St3gal4 WTGGTCGAAGTGGGCCGACTCAGGG 193 KO  +1 bp GGTCGAAGTGGGCCGACcTCA GGG 194KO St3gal6 WT GGAGTTGTGATCATTGTGAGCGG 195 KO  +1 bpGGAGTTGTGATCATTGTtGAG CGG 196 KI ST6AL1 FAB534F1 KO Sort1 WTTTAACAGCAGAGGTATCTGGGGG 197 KO  +1 bp TTAACAGCAGAGGTATCcTGG GGG 198FAB535C2 KO Lrp2 WT TCACACAAGGAATTCCAGTGTGG 199 KO  +1 bpTCACACAAGGAATTCCAaGTG TGG 200 FAB540E9 KO Fut8 WTGATCCGTCCACAACCTTGGCTGG 201 KO  +2 bp GATCCGTCCACAACCTTggGGC TGG 202FAB546A2 KO Gnptab WT GTCACATTCATCGCATCGAGGGG 203 KO  +1 bpGTCACATTCATCGCATCcGAG GGG 204 KO Gnptg WT GCGATGGCGGTGCGGGTGGCCGG 205 KO +1 bp GCGATGGCGGTGCGGGTtGGC CGG 206 FAB555E6 KO Furin WTGACCAAGCGGGACGTGTATCAGG 207 KO  +1 bp GACCAAGCGGGACGTGTtATC AGG 208FAB560A4 KO Manea WT ATAGCCAAGAACTATCCACAAGG 209 KO  +1 bpATAGCCAAGAACTATCCcACA AGG 210 FAB567H3 KO Mgat4b WTGAGAGGCAGGCGCTGCGGGACGG 211 KO  +1 bp GAGAGGCAGGCGCTGCGgGGA CGG 212KO Mgat5 WT GACAATCTCGTCAATGGCACAGG 213 KO  -1 bp GACAATCTCGTCAATG CACAGG 214 KO Gnptab WT GTCACATTCATCGCATCGAGGGG 215 KO  +1 bpGTCACATTCATCGCATCcGAG GGG 216 KO Gnptg WT GCGATGGCGGTGCGGGTGGCCGG 217 KO +1 bp GCGATGGCGGTGCGGGTtGGC CGG 218 FAB568A8 KO Mgat1 WTGAGGGGGTCGCAGGCACACGGGG 219 KO  +1 bp GAGGGGGTCGCAGGCACcACG GGG 220KO Gnptab WT GTCACATTCATCGCATCGAGGGG 221 KO  +1 bp GTCACATTCATCGCATCcGAGGGG 222 KO Gnptg WT GCGATGGCGGTGCGGGTGGCCGG 223 KO  +1 bpGCGATGGCGGTGCGGGTtGGCGGG 224 FAB570B7 KO Mgat2 WTGCGACCGGTACCGCAGCGTTAGG 225 KO  +1 bp GCGACCGGTACCGCAGCcGTT AGG 226KO Gnptab WT GTCACATTCATCGCATCGAGGGG 227 KO  +1 bp GTCACATTCATCGCATCcGAGGGG 228 KO Gnptg WT GCGATGGCGGTGCGGGTGGCCGG 229 KO  +1 bpGCGATGGCGGTGCGGGTtGGC CGG 230 FAB571C2 KO B4galt1 WTGGGCGGTCGTTATTCCCCCAAGG 231 KO  +2 bp GGGCGGTCGTTATTCCCcgCCA AGG 232KO B4galt3 WT GCAGGACGGTACCGGCCCCCTGG 233 KO  -2 bp GCAGGACGGTACCGG

CCC TGG 234 KO Mgat4b WT GAGAGGCAGGCGCTGCGGGACGG 235 KO  +1 bpGAGAGGCAGGCGCTGCGgGGA CGG 236 KO Mgat5 WT GACAATCTCGTCAATGGCACAGG 237 KO -1 bp GACAATCTCGTCAATG CAC AGG 238 KO Gnptab WT GTCACATTCATCGCATCGAGGGG239 KO  +1 bp GTCACATTCATCGCATCcGAG GGG 240 KO Gnptg WTGCGATGGCGGTGCGGGTGGCCGG 241 KO  +1 bp GCGATGGCGGTGCGGGTtGGC CGG 242FAB572E8 KO Ganab WT GAAGGCTTCGATCCTCTAGCAGG 243 KO  +1 bpGAAGGCTTCGATCCTCTtAGC AGG 244 FAB583E4 KO Mgat4b WTGAGAGGCAGGCGCTGCGGGACGG 245 KO  +1 bp GAGAGGCAGGCGCTGCGgGGA CGG 246KO Mgat5 WT GACAATCTCGTCAATGGCACAGG 247 KO  -1 bp GACAATCTCGTCAATG CACAGG 248 KO Gnptab WT GTCACATTCATCGCATCGAGGGG 249 KO  +1 bpGTCACATTCATCGCATCcGAG GGG 250 KO Gnptg WT GCGATGGCGGTGCGGGTGGCCGG 251 KO +1 bp GCGATGGCGGTGCGGGTtGGC CGG 252 KI ST3GAL4 FAB584G10 KO Fut8 WTGATCCGTCCACAACCTTGGCTGG 253 KO  -2 bp GATCCGTCCACACC

GGC TGG 254 KO Mgat4b WT GAGAGGCAGGCGCTGCGGGACGG 255 KO  +1 bpGAGAGGCAGGCGCTGCGgGGA CGG 256 KO Mgat5 WT GACAATCTCGTCAATGGCACAGG 257 KO -1 bp GACAATCTCGTCAATG CAC AGG 258 KO Gnptab WT GTCACATTCATCGCATCGAGGGG259 KO  +1 bp GTCACATTCATCGCATCcGAG GGG 260 KO Gnptg WTGCGATGGCGGTGCGGGTGGCCGG 261 KO  +1 bp GCGATGGCGGTGCGGGTtGGC CGG 262FAB604F9 KO Fam20c WT GGGAAGCCTGACCAGATCGAAGG 263 KO  -4 bpGGGAAGCCTGACCA

GA AGG 264 FAB605D1 KO Golph3 WT GAAAGGCTCAGTGCAACACTGGG 265 KO  +1 bpGAAAGGCTCAGTGCAACcACT GGG 266 FAB606E12 KO Golph3l WTTGACTTCAGTTCGACGGGTACGG 267 KO  +1 bp TGACTTCAGTTCGACGGGgTA CGG 268FAB662F5 KO Alg8 WT TCGGTGTACTTCAAAATCCGTGG 269 KO  +1 bpTCGGTGTACTTCAAAATtCCG TGG 270 FAB664B2 KO Alg12 WTAAATCACCAGGCAAGTCAGGCGG 271 KO  +1 bp AAATCACCAGGCAAGTCcAGG CGG 272FAB667E12 KO Alg5 WT GGACTCTAAGTTCACTTACGAGG 273 KO  -1 bpGGACTCTAAGTTCACT

ACG AGG 274 FAB677F4/C4 KI GNPTG FAB688F7 KO B4galt1 WTGGGCGGTCGTTATTCCCCCAAGG 275 KO −22 bp GG

TC-

- 276 GTATTT KO B4galt3 WT GCAGGACGGTACCGGCCCCCTGG 277 KO-alle1 —ACATCC-

- 278 TTCCGC KO-alle2 −16 bp GCAGGACGGTACC- 279

-CC FAB695G2/A8 KI GNPTAB FAB712D1 KO Man2a1 WT GAGTGAAGCCTCGATCGGGTTGG280 KO  +1 bp GAGTGAAGCCTCGATCGgGGT TGG 281 KO Man2a2 WTGCCCAGAGAAAGCGTCGTCGAGG 282 KO-alle1  +1 bp GCCCAGAGAAAGCGTCGgTCG AGG283 KO-alle2  +2 bp GCCCAGAGAAAGCGTCGGcTCG AGG 284 FAB713A7 KO Man2a1 WTGAGTGAAGCCTCGATCGGGTTGG 285 KO  -2 bp GAGTGAAGCCTCGATC

GT TGG 286 KO Man2a2 WT GCCCAGAGAAAGCGTCGTCGAGG 287 KO  +1 bpGCCCAGAGAAAGCGTCGgTCG AGG 288 KO Gnptab WT GTCACATTCATCGCATCGAGGGG 289KO  +1 bp GTCACATTCATCGCATCcGAG GGG 290 FAB725G5 KO Man1a1 WTGTAAATATACGCTTCGTCGGTGG 291 KO  -2 bp GTAAATATACGCTT TCGG TGG 292KO Man1a2 WT GTCTGTGTTCGTCGGGTCCATGG 293 KO-alle1  +1 bpGTCTGTGTTCGTCGGGTTCCA TGG 294 KO-alle2 −13 bp GTCTGTGTTCGT- 295

-CAG KO Man1b1 WT GAGTACATACCACCTATCGGGGG 296 KO  +1 bpGAGTACATACCACCTATtCGG GGG 297 KO Man1c1 WT GCCCCGGGCGAGGACGATCCCGG 298KO-alle1 +1 bp GCCCCGGGCGAGGACGAaTCC CGG 299 KO-alle2 −17 bp G

CC CGG 300 FAB791F5 KO M6pr WT GCTATAGATTCAGAGTATGCCGG 301 KO  +1 bpGCTATAGATTCAGAGTAaTGC CGG 302 FAB792G6 KO Alg3 WTGCTGCTGGGCTGCGGAAACGCGG 303 KO  +1 bp GCTGCTGGGCTGCGGAAaACG CGG 304KI GNPTAB FAB793F10 KO Alg3 WT GCTGCTGGGCTGCGGAAACGCGG 305 KO  +1 bpGCTGCTGGGCTGCGGAAaACG CGG 306 KI GNPTAB FAB819F7/F6 KI GNPTAB/G FAB857D2KO St3gal4 WT GGTCGAAGTGGGCCGACTCAGGG 307 KO  +1 bpGGTCGAAGTGGGCCGACcTCA GGG 308 KO St3gal6 WT GGAGTTGTGATCATTGTGAGCGG 309KO  +1 bp GGAGTTGTGATCATTGTtGAG CGG 310 KI ST6GAL1 KO Gnptab WTGTCACATTCATCGCATCGAGGGG 311 KO  +1 bp GTCACATTCATCGCATCcGAG GGG 312FAB870B1 KO St3gal4 WT GGTCGAAGTGGGCCGACTCAGGG 313 KO  +1 bpGGTCGAAGTGGGCCGACcTCA GGG 314 KO St3gal6 WT GGAGTTGTGATCATTGTGAGCGG 315KO  +1 bp GGAGTTGTGATCATTGTtGAG CGG 316 KI ST6GAL1 KO Gnptab WTGTCACATTCATCGCATCGAGGGG 317 KO  +1 bp GTCACATTCATCGCATCcGAG GGG 318KO Fut8 WT GATCCGTCCACAACCTTGGCTGG 319 KO  +1 bp GATCCGTCCACAACCTTtGGCTGG 320 NOTE: Nucleic acids UNDERLINED are the gRNA targeting sequence,Nucleic acids in BOLD and UNDERLINED are the PAM sequence, Nucleic acidsin lower case letters are insertions, Nucleic acids in BOLD and italicsare deletions.

We used site-specific glycoprofiling of the secreted purified GLA tomonitor effects on glycosylation. GLA has three N-glycosites (N108,N161, N184), and when expressed in WT CHO cells GLA wassite-specifically glycosylated with mainly complex structures cappedwith SA at N108 and with high-mannose-type M6P glycans at N161 and N184(FIG. 12 a and FIG. 17 , Panels 1-3), in agreement with previousreports. We targeted 43 genes individually or in rational combinationsguided by the sequential biosynthetic pathway of N-glycans and knowngroups of isoenzymes with overlapping functions (Supplementary Table 2and 3). FIG. 11 presents a summary of the observed general trend effectsof the screen for SA, M6P and Man, which are the most importantparameters known to affect biodistribution of ERTs. The specific effectsof each gene targeting on the glycosylation at individual N-glycositesin GLA are shown in FIG. 17 .

Targeting the lipid-linked oligosaccharide precursor assembly on thecytosolic side (Alg1/2/11/13/14) was not successful since viable cellswith bi-allelic KO could not be established in agreement with similarobservations in yeast, however, targeting the precursor assembly on theER luminal side (Alg3/5/6/8/9/12) produced surprising options forsite-specific engineering of M6P-tagging of GLA. KO of Alg3substantially enhanced M6P-tagging at N108, while reducing M6P at N161(FIG. 12 b and FIG. 17 , Panels 4, 5). KO of Alg9 reduced M6P at N161and increased tagging at N184 (FIG. 12 c and FIG. 17 , Panel 6). KO ofAlg12 reduced M6P at N161 and increased M6P at N184 (FIG. 12 d and FIG.17 , Panel 7). KO of Alg6 and Alg8 enhanced hybrid structures with onebranch capped by SA and one with M6P at N161 (FIG. 12 e , FIG. 12 f andFIG. 17 , Panels 9, 10). KO of cis-Golgi mannosidases(Man1a1/1a2/1b1/1c1) enriched oligomannose structures and enhanced M6Pat all three glycosites (FIG. 12 g and FIG. 17 , Panels 12-16), which issupported by previous studies. KO of medial Golgi mannosidase (Man2al/2)created hybrid N-glycans with one branch capped by SA and one witholigomannose at the expense of reduced M6P (FIG. 12 h and FIG. 17 ,Panel 17). KO of Mgat1 as expected completely eliminated complexN-glycans, and interestingly enhanced M6P-tagging at the normal sites,N161 and N184, without inducing M6P-tagging at N108 (FIG. 12 i and FIG.17 , Panel 18). KO of Mgat2 produced the mono-antennary hybrid-typeN-glycan at N108 without affecting M6P at N161 and N184 (FIG. 12 j andFIG. 17 , Panel 19), while KO of Mgat4b/5 completely eliminated tri andtetra antennary N-glycans and increased homogeneity (FIG. 12 k and FIG.17 , Panel 20). The results demonstrate how the content and position ofM6P and exposed Man on lysosomal enzymes can be fine-tuned in greatdetail by gene engineering of CHO cells. Targeting the N-glycan ERglucosidases (Mogs/Ganab) to probe the role of the Glc residues andchaperone interactions, did not affect secretion of GLA substantially(FIG. 18 ), and demonstrated that GLA glycoforms with retained Glcresidues and M6P-tagging can be produced (FIG. 12 l , FIG. 12 m and FIG.17 , Panels 21, 22).

We also targeted the M6P-tagging process. KO of Gnptab or Gnptg of theGlcNAc-1-phosphotransferase complex enabled production of GLA withrather homogeneous complex N-glycans capped by SA at all N-glycositesbut lacking M6P residues (FIG. 12 n , FIG. 12 o and FIG. 17 , Panels 23,24). In addition, KO of the GlcNAc-1-phosphate hydrolase (Nagpa)uncovering enzyme resulted in GLA with GlcNAc residues retained on M6P(M6P-GlcNAc) and interestingly increased M6P-tagging includingsubstantial increase in two M6P tags (bis-M6P) (FIG. 12 p and FIG. 17 ,Panel 26). In addition to two high affinity M6P binding sites, the largecation-independent mannose 6-phosphate receptor (CI-MPR) containsanother preferential binding site for M6P-GlcNAc, but how suchglycoforms would circulate and interact with other receptors is unknown.Targeting the M6P-tagging process may also affect lysosomal targeting ofsome endogenous CHO cell lysosomal enzymes, and resulting changes insecreted lysosomal glycosylhydrolases like e.g. neuraminidase 1 (Neu1)may affect glycan structures of recombinant expressed enzymes.

To explore exposure of GIcNAc we targeted the galactosylation process bydouble KO of B4galt1/3, which substantially reduced galactosylation andresulted in exposed terminal GlcNAc residues on complex N-glycansprimarily at N108 (FIG. 12 q and FIG. 17 , Panel 31). Targeting thesialylation by double KO of St3gal4/6 substantially reduced sialic acidcapping and resulted in exposure of terminal Gal residues (FIG. 12 r andFIG. 17 , Panel 32). Furthermore, targeting the core fucosylation by KOof Fut8 eliminated core fucose without affecting other features (FIG. 12s and FIG. 17 , Panel 33).

We also targeted the genes encoding the M6P receptors CI-MPR (Igf2r) andCD-MPR (M6pr), which may bias the pool of secreted GLA by directinghigh-affinity glycoforms to the lysosome, and while this did notsubstantially affect glycosylation of the secreted GLA, KO of Igf2rslightly increased bis-M6P-tagging at the N184 glycosite (FIG. 17 ,Panels 34, 35). Targeting the late-acting signal peptidase, Sppl3, shownto play a role in shedding of glycosyltransferases45, induced a slightincrease of exposed Man (FIG. 17 , Panel 38). KO of Furin that isimportant for activation of Nagpa46, resulted in similar N-glycanprofile with accumulation of GlcNAc-1-P residues as found with KO ofNagpa confirming the essential role of furin-mediated proproteinactivation of this enzyme (FIG. 12 t and FIG. 17 , Panel 39). Incontrast, targeting the phosphokinase Fam20c and thephosphatidylinositol-4-phosphate (PI4P) effector Golph3 and Golgiprotein Golph3l, did not substantially affect the N-glycosylation of GLA(FIG. 17 , Panels 40-42).

Combinatorial Glycoengineering

The individual gene KO screen provides a matrix for design ofcombinatorial engineering to produce GLA with a wider range of desirableglycoforms known to affect cellular targeting receptors. We firstexplored designs of glycoforms without M6P-tagging. Stacking KO ofGnptab/g with KO of Mgat1 enabled production of GLA with high mannoseN-glycans at all three glycosites (4-5 Man residues) (FIG. 13 a and FIG.17 , Panel 44). Such high-Man glycoforms have been shown to bind MRexpressed on macrophages and efficiently target the liver and spleen.Stacking KO of Man2al/2 involved in the α-mannosidase trimming processof the a6-branch on top of Gnptab KO generated GLA with a mono-antennaryhybrid structure with a complex sialylated a3-arm combined with threeMan residues on the a6-arm (FIG. 13 b and FIG. 17 , Panel 45), which mayalter binding to both MPRs and MR. Similarly, stacking with KO of Mgat2enables production of GLA with mono-antennary hybrid N-glycan, but witha single Man residue at the a6-arm that may even further reduce MRbinding (FIG. 13 c and FIG. 17 , Panel 46). This design was lesshomogeneous due to branching at the a3-arm, however, stacking KO ofGnptab/g with KO of Mgat4b/5 enables production of GLA with homogeneousbi-antennary complex N-glycans with SA capping (FIG. 13 d and FIG. 17 ,Panel 47), and this would also apply with the Mgat2 KO design. Theseglycoforms are all expected to have no interaction with the MPRs, andinstead enable fine-tuning of interactions with the MR guided by thedifferent exposure of terminal Man and content of SA capped glycans.

Next, we focused on improving M6P-tagging by first testing individualknock-in (KI) of GNPTG or GNPTAB, which enhanced M6P at N161 and N184,and with KI of GNPIAB induced bis-M6P at N184 (FIG. 13 e,f and FIG. 17 ,Panels 48-51). We used targeted KI with Zinc-finger nucleases (ZFNs)(modified ObLiGaRe strategy, which is described in Maresca, M., Lin, V.G., Guo, N. & Yang, Y. Obligate ligation-gated recombination (ObLiGaRe):customdesigned nuclease-mediated targeted integration throughnonhomologous end joining. Genome Res 23, 539-546 (2013), incorporatedherein by reference in its entirety) or CRISPR/Cas9 facilitatednon-homologous end-joining into a CHO Safe-Harbor locus (as described inGeisinger, J. M., Turan, S., Hernandez, S., Spector, L. P. & Calos, M.P. In vivo blunt-end cloning through CRISPR/Cas9-facilitatednon-homologous end-joining. Nucleic Acids Res 44, e76 (2016); Yang, Z.et al. Engineered CHO cells for production of diverse, homogeneousglycoproteins. Nat Biotechnol 33, 842-844 (2015); and Bahr, S., Cortner,L., Ladley, S. & Borgschulte, T. in BMC proceedings, Vol. 7 P3 (BioMedCentral, 2013), each of which is incorporated herein by reference in itsentirety). Moreover, combined KI of both genes induced a substantialincrease in M6P-tagging at all three glycosites and with high content ofthe mono-antennary hybrid structure with SA and M6P (FIG. 13 g and FIG.17 , Panels 52-53). KI of GNPTAB combined with KO of Alg3 enabledproduction of a unique high-Man N-glycan with efficient M6P-tagging atall N-glycosites exclusively on the a3-arm (FIG. 13 h and FIG. 17 ,Panels 54, 55). The CI-MPR has multiple binding sites and the capacityto bind diverse M6P-tagged structures with different affinities⁵³, andincreasing the M6P content and introducing bis-M6P are predicted toenhance uptake as demonstrated e.g. with the acid α-glucosidase (GAA)used for ERT of Pompe disease.

We also engineered cells to produce GLA with homogenous α2-6SA capping.CHO WT cells only have capacity for α2-3SA capping, and systematicstudies of the influence of α2-3SA versus α2-6SA capping found on mosthuman serum glycoproteins have not been performed in the past, althoughthe interaction with many receptors are affected by the linkage of theSA, including e.g. Galectins that are blocked by α2-6SA and Siglecs thatexhibit differential interactions with the different SAs. The AMR mayhave different interactions with glycans capped by α2-6SA. Stacked KO ofGnptab and St3gal4/6 with targeted KI of ST6GAL1 enabled production ofGLA with homogeneous α2-6SA capping and higher content of bi-antennarystructures (FIG. 13 i and FIG. 17 , Panel 58). KO of Mgat4b/S with KI ofST3GAL4 enabled production of homogenous biantennary N-glycans cappedwith α2-3SA (FIG. 13 j and FIG. 17 , Panel 59). Combined with KO ofFut8, any glycoform may likely be produced without core fucose (FIG. 13k,l and FIG. 17 , Panels 60, 61).

The Glycoengineering Matrix is Applicable to Other ERTs

In order to explore the extent to which the glycoengineering designs areapplicable to other lysosomal enzymes, we stably expressed human GBA inCHO WT and tested representative engineering designs (FIG. 14 and FIG.19 and Supplementary Tables 4 and 5).

TABLE 8 Summary of CHO mutant clones stably expressing GBA and cell lineancestry. Project Parental CHO number Targeted genes line GBA826 KOGnptab GBA#A5 GBA827 KO Alg3 GBA#A5 GBA828 KO Mgat1 GBA#A5 GBA829 KOAlg9 GBA#A5 GBA831 KO Gnptab/Mgat1 GBA#A5 GBA900 KO Gnptab/Man2a1/2GBA826B1 GBA901 KO Gnptab/Mgat2 GBA826B1

TABLE 9 Sequence analysis of CHO mutant clones stably expressing GBA.Clone Targeted genes InDels Alignment SEQ ID NO: GBA826B1 KO Gnptab WTGTCACATTCATCGCATCGAGGGG 321 KO +1 bp GTCACATTCATCGCATCCGAG GGG 322GBA827C2 KO Alg3 WT GCTGCTGGGCTGCGGAAACGCGG 323 KO −1 bpGCTGCTGGGCTGCGGA

ACG CGG 324 GBA827D2 KO Alg3 WT GCTGCTGGGCTGCGGAAACGCGG 325 KO +1 bpGCTGCTGGGCTGCGGAACACG CGG 326 GBA828D9 KO Mgat1 WTGAGGGGGTCGCAGGCACACGGGG 327 KO +1 bp GAGGGGGTCGCAGGCACCACG GGG 328GBA828E9 KO Mgat1 WT GAGGGGGTCGCAGGCACACGGGG 329 KO +1bpGAGGGGGTCGCAGGCACcACG GGG 330 GBA829F2 KO Alg9 WTGAGCAGACATTTGAAAGCAGTGG 331 KO-alle1 −7 bp GAGCAGACAT

GCAG TGG 332 KO-2alle2 −2 bp GAGCAGACATTTGAtttG TGG 333 GBA831C8KO Mgat1 WT GAGGGGGTCGCAGGCACACGGGG 334 KO +1 bp GAGGGGGTCGCAGGCACcACGGGG 335 KO Gnptab WT GTCACATTCATCGCATCGAGGGG 336 KO +1 bpGTCACATTCATCGCATCcGAG GGG 337 G3A831F10 KO Mgat1 WTGAGGGGGTCGCAGGCACACGGGG 338 KO +1 bp GAGGGGGTCGCAGGCACcACG GGG 339KO Gnptab WT GTCACATTCATCGCATCGAGGGG 340 KO +1 bp GTCACATTCATCGCATCcGAGGGG 341 GBA900D6 KO Man2a1 WT GAGTGAAGCCTCGATCGGGTTGG 342 KO −4 bpGAGTGAAGCCTCG

GGT TGG 343 KO Man2a2 WT GCCCAGAGAAAGCGTCGTCGAGG 344 KO −1 bpGCCCAGAGAAAGCGTCG

CG AGG 345 KO Gnptab WT GTCACATTCATCGCATCGAGGGG 346 KO +1 bpGTCACATTCATCGCATCcGAG GGG 347 GBA901D9 KO Mgat2 WTGCGACCGGTACCGCAGCGTTAGG 348 KO +1 bp GCGACCGGTACCGCAGCcGTT AGG 349KO Gnptab WT GTCACATTCATCGCATCGAGGGG 350 KO −1 bp GTCACATTCATCGCATCcGAGGGG 351 NOTE: Nucleic acids UNDERLINED are the gRNA targeting sequence,Nucleic acids in BOLD and UNDERLINED are the PAM sequence, Nucleic acidsin lower case letters are insertions, Nucleic acids in BOLD and italicsare deletions.

GBA has 4 N-glycan sites of which N19 and N59 are mainly occupied bycomplex biantennary N-glycans, while those at N146 and N270 contain amixture of complex bi-, tri- and tetraantennary N-glycans and M6P-taggedN-glycans, with N270 being the major M6P-tagged glycosite (FIG. 14 a andFIG. 19 , Panel 1). This is in agreement with previous reports. KO ofAlg3 increased the M6P content in general and in particular for the N146and N270 glycans (FIG. 14 b and FIG. 19 , Panels 2). KO of Alg9 hadlittle effect on the N-glycans at N19 and N59, but altered theoligomannose structures with M6P at N146 and N270 (FIG. 14 c and FIG. 19, Panel 3). Targeting Gnptab resulted in rather homogeneous complex typeN-glycans with SA capping at all four N-glycosites and no M6P content(FIG. 14 d and FIG. 19 , Panel 4). Targeting Mgat1 enabled production ofGBA without complex type N-glycans, but with high mannose glycans andreduced M6P mainly at N270 (FIG. 14 e and FIG. 19 , Panels 5, 6).Stacked KO of Gnptab and Mgat1 enabled production of GBA with highmannose N-glycans without M6P at all glycosites and oligomannosestructures consisting of 4-5 Man residues (FIG. 14 f and FIG. 19 ,Panels 7, 8). Stacked KO of Man2al/2 and Gnptab generated GBA with arather homogeneous mono-antennary hybrid structure at all 4 glycositeswith a complex sialylated a3-arm combined with three Man residues on thea6-arm (FIG. 14 g and FIG. 19 , Panel 9). Similarly, GBA withmono-antennary hybrid N-glycan carrying a single Man residue at thea6-arm was generated by stacking KO of Mgat2 and Gnptab (FIG. 14 h andFIG. 19 , Panel 10). These two designs may represent a glycodesign withlower MR binding and increased circulation. In general, the outcome ofthe engineering performed with GBA correlated well with the effectsobserved with GLA, when considering the inherent site-specificity ofN-glycan processing found with the enzymes expressed in WT CHO cells. Wepredict that further studies with KI of GNPTAB and GNTPG may induceM6P-tagging at all N-glycosites similar to our findings for GLA and asreported previously.

Analyses of GLA Glycoforms in a Deficient Fabry Disease Mouse Model

Fabry disease is caused by deficiency in GLA and the leading ERT isFabrazyme (GenZyme) produced in CHO cells. We first benchmarked GLAproduced in our CHO WT cell (100 mgs/L) with a clinical lot of Fabrazyme(GenZyme) finding lower content of exposed Man residues on GLA producedby us (FIG. 15A and FIG. 17 , Panels 1, 3). The two CHO WT produced GLAexhibited similar blood circulation half-time (FIG. 15B) with trends ofhigher liver targeting and lower spleen, kidney and heart targeting ofour GLA, although only the lower kidney targeting was significant (FIG.15D).

We then tested five engineered distinct glycoforms of GLA in directcomparison with Fabrazyme at 1 mg/kg dose (FIG. 15A). The engineeringdesign and detailed structure analysis of the selected glycovariants areshown in FIG. 17 , Panels 1, 6, 55, 52, 59 and 58). The specificactivity and stability in plasma of these GLA glycovariants wereessentially identical (FIG. 21 ). Three glycoforms designed withslightly lower M6P (LoM6P), higher M6P (HiM6P), or higher M6P contentwith mainly the hybrid-type (HybM6P) produced trends towards higher orlower circulation time with half-lifes of 15.4±±1.1 min, 11.0±2.0 min,and 8.3±0.8 min, respectively, compared with 9.8±0.3 min for Fabrazyme(FIG. 15C). These three glycoforms showed minor differences in targetingto select organs compared to Fabrazyme with the LoM6P glycoform yieldingsignificantly higher levels of enzyme activity in the heart and HiM6Pand HybM6P exhibiting lower levels in spleen and liver (FIG. 15E). Thesetrends are consistent with MPR-mediated uptake, and the relatively highlevel of M6P content of Fabrazyme likely influence the degree ofdifferences.

In striking contrast, the two glycoforms designed with N-glycans cappedby sialic acids and without M6P and exposed Man, produced significantchanges in circulation and biodistribution (FIGS. 15C and 15E).GLA-Bi23SA with homogeneous biantennary N-glycans capped with α2-3SA(FIG. 15A), exhibited a markedly extended (3-fold) circulation time(half-life 27.5±0.8 min) (FIG. 15C), and lower enzyme activity in liver,spleen, and kidney, but the highest level of enzyme in the heart amongall glycoforms tested (FIG. 15E). Importantly, the GLA-LoM6P showed thesame trend as would be predicted. The most frequent cause of death inpatients with Fabry disease is cardiomyopathy, and increased delivery tothe cardiovascular system with glycoforms such as GLA-Bi23SA may presenta promising solution. The impact of the 3-fold increase in circulationtime of GLA-Bi23SA should be viewed in light of the finding that GLA hasextremely poor stability in plasma at 37° C. with loss of more than 50%activity within 15 min (Kizhner, T. et al. Characterization of achemically modified plant cell culture expressed humanalpha-Galactosidase-A enzyme for treatment of Fabry disease. Mol GenetMetab 114, 259-267 (2015); Sakuraba, H. et al. Comparison of the effectsof agalsidase alfa and agalsidase beta on cultured human Fabryfibroblasts and Fabry mice. J Hum Genet 51, 180-188 (2006), each ofwhich is incorporated herein by reference in its entirety). A PEGylatedversion of GLA (PRX-102) only enhanced the stability in plasmaapproximately 2-fold (Kizhner 2015).

GLA-26SA was designed to have N-glycans capped with α2-6SA (FIG. 15A),and perhaps surprisingly (see Park, E. I., Mi, Y., Unverzagt, C.,Gabius, H. J. & Baenziger, J. U. The asialoglycoprotein receptor clearsglycoconjugates terminating with sialic acid alpha 2,6GalNAc. Proc NatlAcad Sci USA 102, 17125-17129 (2005), incorporated herein by referencein its entirety), this glycoform resulted in markedly increased liveruptake and corresponding decrease in spleen and kidney uptake (FIG.15E), and the circulation time was only marginally elevated (FIG. 15C).The GLA-26SA glycoform produced the lowest level of enzyme activity inthe kidney (FIG. 15E). The striking increase in liver uptake of theα2-6SA capped glycoform resembles previous studies obtained with analbumin neoglyconjugate suggesting interaction with the AMR, but severaltherapeutic glycoproteins produced in human cells including HEK293 havepartial α2-6SA capping and appear to function similar to those producedin CHO cells with only α2-3SA.

Cellular localization of Fabrazyme and the glycovariants in the heart,kidney and liver was assessed by immunohistochemistry (FIG. 15F). Thelocalization pattern ofFabrazyme in these organs was consistent withthat of agalsidase alfa reported in previous studies. In the heart,Fabrazyme and all five glycovariants were detected in vascular and/orperivascular cells, but not in cardiomyocytes (FIG. 15F). There were noclear differences between the tested variants. In the kidney, Fabrazymeand GLA-LoM6P, GLA-HiM6P, GLA-HybM6P, and GLA-Bi23SA were predominantlydetected in tubular epithelial cells. However, GLA-26SA hadsignificantly decreased number and intensity of positive signals intubules compared to the other variants tested (FIG. 15F). In the liver,Fabrazyme, GLA-LoM6P, GLA-HiM6P, and GLA-HybM6P were detected inhepatocytes, putative Kupffer cells and endothelial cells of sinusoidalcapillaries. GLA-Bi23SA was also detected in these cell types; however,the number of positive signals in hepatocytes was clearly decreasedcompared to Fabrazyme. Distribution of GLA-26SA in the liver wasremarkably different from other variants; This variant was detectedalmost exclusively in hepatocytes, and the number of positive signals inhepatocytes was clearly increased compared to Fabrazyme (FIG. 15F).

Encouraged by the unique performance of GLA-Bi23SA we also tested theeffect of this glycoform on reduction of accumulatedglobotriosylceramide (Gb3) substrate in organs 2 weeks after a singleinjection of 1 mg/kg. GLA-Bi23SA treatment resulted in a similarreduction of the Gb3 content in heart, kidney and liver as compared toFabrazyme (FIG. 15G). This unequivocally demonstrate that glycoforms ofGLA without the classical receptor ligands M6P and Man are efficientlytaken up by cells, delivered to the lysosome, and functioning inreduction of the Gb3 substrate accumulation.

In summary, the glycoengineered GLA variants exhibited distinctpharmacodynamic profiles in Fabry mice. The α2-3SA sialylated designwithout M6P and terminal Man led to reduced uptake by hepatocytes,prolonged plasma half-life, and improved delivery to the heart. Incontrast, the α2-6SA sialylated design led to preferential delivery tohepatocytes and decreased uptake by renal tubular cells. The longercirculation time of GLA-Bi23SA is likely to provide opportunity forwider organ distribution as evidenced by the markedly increase in uptakein the heart, which is critical for many LSDs. Longer circulation timemay also provide opportunity for use of lower enzyme dose or applicationfrequency of replacement enzymes, although further studies are needed toaddress this.

DISCUSSION

The comprehensive engineering performed with GLA and GBA in CHO cellsdemonstrates that there are wide options for fine-tuning all keyfeatures of N-glycans on lysosomal enzymes known to be important fortheir cellular uptake, biodistribution and bioavailability. Thisincludes a high degree of site-specific fine-tuning of M6Pstoichiometry, exposure of Man, Gal, and GlcNAc residues, and capping bySA. We provide novel designs for recombinant lysosomal enzymes whichlack recognition markers for classical MPRs and MRs but containinghomogenous N-glycans capped by SA. Among these we discover GLA-Bi23SAthat offer increased circulation time, efficient cellular uptake andimproved organ distribution following its application in mice. Thissuggests that the α2-3SA design can be used to overcome one of thearguably major obstacle for many ERTs, i.e. their rapid clearance fromcirculation by liver and spleen. Extended circulation is predicted toenable wider bioavailability and possibly transport across theblood-brain-barrier (Damme, M. et al. Chronic enzyme replacement therapyameliorates neuropathology in alphamannosidosis mice. Ann Clin TranslNeurol 2, 987-1001 (2015), incorporated herein by reference in itsentirety). The achieved control of N-glycosylation in CHO cells meets orsurpass the glycoengineering opportunities previously presented withnon-mammalian cells and postproduction modification strategies. Theclinical features of LSDs and the organs affected differ greatly as dothe biostructural properties of the respective deficient enzymes, andthe design matrix and glycoengineered CHO cells developed here will bevaluable tools for production and testing of optimal designs forindividual ERTs, in order to improve a class of essential drugs withhigh costs and poor performance.

CHO cells are the preferred mammalian expression hosts for humantherapeutics. Given the recent options for targeted and stable geneengineering of glycosylation capacities in mammalian cells, we undertookto explore the glycoengineering options for M6P-modified lysosomalreplacement enzymes that represent one of the most complex challengesfor the biopharmaceutical industry (Platt, F. M. Emptying the stores:lysosomal diseases and therapeutic strategies. Nat Rev Drug Discov 17,133-150 (2018); Parenti, G., Pignata, C., Vajro, P. & Salerno, M. Newstrategies for the treatment of lysosomal storage diseases (review). IntJ Mol Med 31, 11-20 (2013); Parenti, G., Andria, G. & Ballabio, A.Lysosomal storage diseases: from pathophysiology to therapy. Annu RevMed 66, 471-486 (2015), each of which is incorporated herein byreference in its entirety). Using GLA as an illustrative example wedissected virtually all steps in the genetic and biosynthetic control ofN-glycosylation and M6P-tagging, and found surprising plasticity andcontrol for fine-tuning complex N-glycan patterns even with a degreeofglycosite specificity (FIGS. 12 and 13 ). Thus, M6P-tagging could betuned up and down and directed to one (N184), two, or all threeN-glycosites of GLA, and importantly combined with different degrees ofhigh-Man or complex sialylated N-glycans. Moreover, it was possible toinduce the hybrid N-glycan with a sialylated a3-arm and an M6P-taggeda6-arm. For the first time we also demonstrated production of glycoformswith homogenous SA capping but lacking M6P or exposed Man residues.

It has long been clear that the structure of N-glycans on replacementenzymes affects cellular uptake and circulation time by interacting withdifferent cell surface receptors, and that altering the glycancomposition can be used to direct organ-targeting. This was demonstratedfirst with targeting of GBA with high-Man structures for the MR onmacrophages, and ERTs with glycans optimized for targeting specificreceptors are already successfully used in the clinic. Differentstrategies have been applied to optimize N-glycans for specific cell andorgan targeting requirements. To achieve N-glycans with high degree ofMan exposure for MR-mediated liver targeting, e.g. for GBA treatment ofGaucher patients, the industry has used plant cells, human fibrosarcomacells combined with N-glycan mannosidase inhibitors, and CHO cellscombined with postproduction treatment with multiple exoglycosidases. Wepresent engineered CHO cells capable of producing this high-Manglycoform of GBA (FIG. 14 f ), and importantly also related designs withdifferent degrees of Man exposure and SA capping expected to influencekinetics of uptake and circulation half-life (FIG. 14 g ). To increasethe M6P content in particular for targeting muscle cells, yeast has beenused to produce the lysosomal α-glucosidase deficient in Pompe disease.Yeast modify human lysosomal enzymes with Man-Pi-6-Man, but the elegantintroduction of an uncovering α-mannosidase enzyme results in productionof α-glucosidase rich in M6P. Other strategies to increase M6P contentinclude in vitro chemical conjugation, or co-expression of a truncatedGlcNAc-1-phosphotransferase a/p precursor. These strategies do notenable fine control over the content (or site-specificity) of M6P andother glycan features including SA capping, and the presentedengineering of high-M6P glycoforms in CHO cells fully match thesestrategies (FIGS. 13 f-13 h ). Other postproduction modificationstrategies including oxidative reduction of glycans and PEGylation havebeen applied to reduce glycan-mediated receptor uptake and/or enhancecirculation, and these may be met by the presented glycoform designswith homogeneous SA capping but lacking M6P or exposed Man residues(FIG. 13 j ). Thus, our study suggests that any of the more complexprocesses used for production of enzymes required for ERTs in the clinictoday or in development, can be produced simpler and more effective inglycoengineered CHO cells. Moreover, there may be advantages incombining distinct glycoforms of lysosomal enzymes with emergingglycosylation-independent targeting strategies developed forblood-brain-barrier transport.

The general factors determining glycan-mediated receptor uptake are thepresence of exposed M6P, Man, Gal, and GlcNAc residues, but ourunderstanding of and ability to predict the outcome of interactionsbetween glycoproteins with heterogeneous N-glycans presenting thesefeatures and the multiple receptors involved is limited. Numerousstudies have explored the binding and uptake of extreme glycoforms suchas high-Man and high/low M6P-containing lysosomal enzymes, butsystematic studies investigating the complex interplay between differentglycan features have not been possible due to lack of methods to producesuch glycoforms. Studies with e.g. the lysosomal alpha-mannosidase(LAMAN) that contains multiple N-glycans with very low M6P-content andexposure of Man when produced in WT CHO cells, suggest that limitedinteraction with the MPRs and MR may be advantageous for widerbiodistribution and crossing into the brain possibly due to extendedcirculation time. Similar findings were observed with postproductionmodified enzymes with partially destroyed glycans. Here, we exploredfive distinct glycoforms of GLA including two lacking M6P or exposed Manresidues in a Fabry disease mouse model, and found significant changesin circulation half-life and biodistribution (FIG. 15 ). Mostsignificantly, the GLA glycoform with α2-3SA capped N-glycans not onlyshowed enhanced circulation time but also demonstrated efficient uptakeand function in all tested organs with improved distribution to thehard-to-reach heart compared to the leading Fabrazyme variant (FIG.15E), as well as to a recent moss-produced high-Man variant. Evaluatingthe relative organ distributions of glycovariants among the four majorvisceral organs tested illustrate the substantial improved distributionof GLA-Bi23SA to the heart and other organs except the liver (FIG. 22 ).The mechanism for uptake of the α2-3SA capped GLA glycoform is notclear, but studies have shown that lysosomal targeting of GLA is atleast partly independent on M6P-tagging, and other endocytic receptorssuch as sortilin (SORT1) and LRP2 (megalin) may be involved. The 3-foldincrease in circulatory half-life for α2-3SA capped GLA is lower thanthe increase observed with e.g. oxidative degradation and reduction ofthe 0-glucuronidase (GUS), but this likely reflects the extremely lowstability of GLA in plasma. It may be interesting to explore combiningthis glycoform with the stabilizing molecular chaperone AT1001(Benjamin, E. R. et al. Co-administration with the pharmacologicalchaperone AT1001 increases recombinant human alpha-galactosidase Atissue uptake and improves substrate reduction in Fabry mice. Mol Ther20, 717-726 (2012); Xu, S. et al. Coformulation of a Novel Humanalpha-Galactosidase A With the Pharmacological Chaperone AT1001 Leads toImproved Substrate Reduction in Fabry Mice. Mol Ther 23, 1169-1181(2015), each of which is incorporated herein by reference in itsentirety) or PEGylation (Kizhner, T., 2015), and also to considertherapeutic modalities comprised of multiple distinct glycoforms.

In summary, the comprehensive CHO glycoengineering performed and thedesign matrix generated for lysosomal enzymes, opens systematic studieson options for improving ERTs by designed glycan features. Past studieshave demonstrated the value of changing the structures of glycans onenzymes needed for ERTs, but the full potential has clearly not been metby use of yeast and plant production platforms or postproductionmodification strategies. The CHO production platform offer new designcapabilities, and the remarkable performance of found for GLA cappedwith SA may represent a new design paradigm for many ERTs.

Methods

Establishment of Stable CHO Clones Expressing Recombinant Human GLA andGBA Enzymes.

An expression construct containing the entire coding sequence of humanGLA was synthesized by Genewiz, USA. Full length cDNA of human GBA1 waspurchased from Sino Biological Inc., China. Both constructs weresubcloned into modified pCGS3 (Merck/formally known as Sigma-Aldrich)for glutamine selection in CHOZN GS−/− cells. CHO cells were maintainedas suspension cultures in serum-free media (EX-CELL CHO CD Fusion, cat.no 14365C), supplemented with 4 mM L-glutamine in 50 mL TPP TubeSpin®Bioreactors with 180 rpm shaking speed at 37° C. and 5% CO₂. Cells wereseeded at 0.5×10⁶ cells/mL in T25 flask (NUNC, Denmark) one day prior totransfection. Electroporation was conducted with 2×10⁶ cells and 8 μgendotoxin-free plasmids using Amaxa kit V and program U24 with AmaxaNucleofector 2B (Lonza, Switzerland). Electroporated cells weresubsequently plated in 6-wells with 3 mL growth media, and after 72 hrscells were plated in 96-wells at 1,000 cells/well in 200 μl MinipoolPlating Medium containing 80% EX CELL@ CHO Cloning Medium (Cat.no C6366)and EX-CELL CHO CD Fusion serum-free media without glutamine. Highexpressing clones were selected by assaying the medium for enzymeactivity (GLA) or with an ELISA using anti-HIS antibodies (for GBA), andselected clones were scaled-up in serum-free media without L-glutaminein 50 mL TPP TubeSpin® shaking Bioreactors (180 rpm, 37° C. and 5% CO₂)for enzyme production.

Purification of GLA and GBA

For GLA spent culture medium was centrifuged at 500×g for 20 min,filtered (0.45 μm), diluted 3-fold with 25 mM MES (pH 6.0), and loadedonto a DEAE-Sepharose Fast Flow column (Sigma). The column was washedwith 10 column volumes (CV) washing buffer (25 mM MES with 50 mM NaCl,pH 6.0) and eluted with 5 CV elution buffer (25 mM MES with 200 mM NaCl,pH 6.0). For mouse studies GLA was further purified by Mono-Qchromatography. For the HIS-tagged GBA culture medium was centrifuged,filtered, and mixed 3:1 (v/v) in 4× binding buffer (200 mM Tris, pH 8.0,1.2 M NaCl) and applied to 0.3 ml packed NiNTA agarose (Invitrogen),pre-equilibrated in binding buffer (50 mM Tris, pH 8.0, 300 mM NaCl).The column was washed with binding buffer and eluted with binding bufferwith 250 mM imidazole. Purity and quantification was evaluated bySDS-PAGE Coomassie staining.

CRISPR/Cas9 targeted KO in CHO cells

Gene targeting was performed in CHO clones stably expressing GLA or GBA.Cells were seeded at 0.5×10⁶ cells/mL in T25 flask (NUNC, Denmark) oneday priorto transfection, and 2×10⁶ cells and 1 μg each of endotoxinfree plasmid DNA of Cas9-GFP and gRNA in the plasmid U6GRNA (AddgenePlasmid #68370) were used for electroporation as described above. 48 hrsafter nucleofection the 10-15% highest labeled (GFP) pool of cells wereenriched by FACS, and after 1 week in culture cells were single cellsorted by FACS into 96-wells. KO clones were identified by IndelDetection by Amplicon Analysis (IDAA) as described (Lonowski, L. A. etal. Genome editing using FACS enrichment of nuclease-expressing cellsand indel detection by amplicon analysis. Nature Protocols 12, 581-603(2017), incorporated herein by reference in its entirety), as well aswhen possible by immunocytology with appropriate lectins or monoclonalantibodies. Selected clones were further verified by Sanger sequencing.The strategy enabled fast screening and selection of KO clones withframeshift mutations, and on average we selected 2-5 clones from eachtargeting event.

ZFNs/CRISPR-Mediated KI in CHO Cells

Site-specific CHO Safe-Harbor locus KI was based on ObLiGaRe strategyand performed with 2 μg of each ZFN (Merck/formerly known asSigma-Aldrich) tagged with GFP/Crimson as previously described (Yang, Z,Nature Biotechnol 33, 2015), and 5 μg donor plasmid with full codinghuman genes (ST3GAL4, ST6GAL1, GNPIAB, or GNPTG). In brief, the EPB69donor plasmid contained inverted CHO Safe-Harbor locus ZFN binding sitesflanking the CMV promoter-ORF-BGH polyA terminator. Mono-allelictargeted KI clones with one intact allele were selected by IDAA analysis(Yang, Z, Nucleic Acids Res 42, 2015). To stack a second gene into aSafe-Harbor locus, we first designed gRNA for the CHO Safe-Harbor locusflanking the ZFN binding site, followed by transfection with 1 μg of adonor PCR product of gene to be inserted with 1 μg Cas9-GFP and 1 μggRNA. In brief, the donor PCR product was generated by using EPB69 donorplasmid as template which contained the CMV promoter-ORF-BGH polyAterminator. KI clones were screened by PCR with primers specific for thejunction area between the donor plasmid and the Safe-Harbor locus. Aprimer set flanking the targeted KI locus was used to characterize theallelic insertion status, and when possible, KI clones were alsoscreened by immunocytology with lectins and monoclonal antibodies.

GLA Enzyme Activity Assay

GLA enzyme activity was measured with 33 mM (unless otherwise specified)p-nitrophenyl-α-D-galactopyranoside (pNP-Gal) at 37° C. for 30 min at pH4.6 in 20 mM citrate and 30 mM sodium phosphate, and the reaction wasquenched with borate buffer (pH 9.8) and released p-nitrophenol was readat 405 nm. A standard curve was generated by using 1:2 serially dilutedp-Nitrophenol in the same assay condition to calculate the amount ofreleased product.

Site-Specific N-Glycopeptide Analysis

Approximately 10 μg of purified GLA or GBA in 50 mM Ammoniumbicarbonatebuffer (pH 7.4) was reduced with dithiothreitol (10 mM) at 60° C. for 30min and alkylated with iodoacetamide (20 mM) for 30 min in dark at roomtemperature. Chymotrypsin digestion was performed at a 1:25 enzyme:substrate ratio. The proteolytic digest was desalted by custom mademodified StageTip columns containing 2 layers of C18 and 1 layer of C8membrane (3M Empore disks, Sigma-Aldrich). Samples were eluted with 50%methanol in 0.1% formic acid, and then dried in SpeedVac andre-solubilized in 0.1% formic acid. LC MS/MS analysis was performed withan EASY-nLC 1000 LC system (ThermoFisher Scientific) interfaced viananoSpray Flex ion source to an Orbitrap Fusion MS (ThermoFisherScientific). Briefly, the nLC was operated in a single analytical columnset up using PicoFrit Emitters (New Objectives, 75 μm inner diameter)custom packed with Reprosil-Pure-AQ C18 phase (Dr. Maisch, 1.9-μmparticle size, 19-21 cm column length). Each sample was injected ontothe column and eluted in a gradient from 2 to 25% B in 45 min at 200nL/min (Solvent A, 100% H₂O; Solvent B, 100% acetonitrile; bothcontaining 0.1% (v/v) formic acid). A precursor MS1 scan (m/z 350-2,000)of intact peptides was acquired in the Orbitrap Fusion at the nominalresolution setting of 120,000, followed by Orbitrap HCD-MS2 and at thenominal resolution setting of 60,000 of the five most abundant multiplycharged precursors in the MS1 spectrum; a minimum MS1 signal thresholdof 50,000 was used for triggering data-dependent fragmentation events.Targeted MS/MS analysis was performed by setting up a targeted MS^(n)(tMS^(n)) Scan Properties pane.

Data Analysis

Glycopeptide compositional analysis was performed from m/z featuresusing in-house written SysBioWare software. For m/z feature recognitionfrom full MS scans LFQ Profiler Node of the Proteome discoverer 2.1(ThermoFisher Scientific) was used. A list of precursor ions (m/z,charge and retention time) was imported as ASCII data into SysBioWareand compositional assignment within 4 ppm mass tolerance was performed.The main building blocks used for the compositional analysis were:NeuAc, Hex, HexNAc, dHex and phosphate. The most prominent peptidescorresponding to each potential glycosites were added as an additionalbuilding block for compositional assignment. The most prominent peptidesequence related to each N-glycosite was determined experimentally bycomparing the yield of deamidated peptides before and after PNGase Ftreatment. A list of potential glycopeptides and glycoforms for eachglycosite was generated and the top 10 of the most abundant candidatesfor each glycosite were selected for targeted MS/MS analysis to confirmthe proposed structure. Each targeted MS/MS spectrum was subjected tomanual interpretation. Same N-glycan composition may represent isobaricstructures, so the listed glycan structure were assisted by and inagreement with literature data, predicted enzyme functions of thetargeted genes together with useful information in MS/MS fragments.

Mouse Studies

Fabry mice (˜3.5 months male) and WT controls were used as reportedpreviously (Shen, J. S. et al. Mannose receptor-mediated delivery ofmoss-made alpha-galactosidase A efficiently corrects enzyme deficiencyin Fabry mice. J Inherit Metab Dis 39, 293-303 (2016), incorporatedherein by reference in its entirety). All animal procedures werereviewed and approved by the Institutional Animal Care and Use Committeeof Baylor Research Institute. All injections were performed via thetail-vein with enzymes diluted in saline to a total volume of 200 μl permouse.

Pharmacokinetics

Enzyme preparations were injected at a dose of 1 mg/kg body weight. Atindicated time points, blood samples were collected from tail vein,plasma was separated by centrifugation, and used for enzyme assay.

Biodistribution and Tissue Kinetics.

Enzyme preparations were injected at a dose of 1 mg/kg body weight. Atindicated time points, mice were perfused with saline (to remove blood),and heart, kidney, liver and spleen were dissected. The whole organswere homogenized in 0.2% Triton/saline for enzyme assay. Proteinconcentration was measured using BCA protein assay kit (Pierce).

Immunohistochemistry

Enzyme preparations were injected at a dose of 2 mg/kg body weight.Heart, kidney and Liver were harvested 24 h after enzyme infusion.Untreated Fabry mouse tissues were used as negative controls. Tissueswere fixed in formalin, embedded in paraffin, and 5-micron sections weremade. IHC was performed by the Histopathology and Tissue Shared Resourcein Georgetown University (Washington, D.C.). In brief, afterheat-induced epitope retrieval in citrate buffer, sections were treatedwith 3% hydrogen peroxide and 10% normal goat serum, and were incubatedwith rabbit polyclonal antibody to human GLA (Shire). After incubationwith HRP-labeled secondary antibody, signals were detected by DABchromogen, and the sections were counterstained with hematoxylin. Signalspecificity was verified with control staining, in which the primaryantibody incubation was omitted. We also developed a mouse monoclonalantibody to purified recombinant human GLA that was used to verify IHC.

Clearance of Tissue Gb3

Enzyme preparations or vehicle alone (saline) were injected into 6months old female Fabry mice at doses of 1 mg/kg body weight. Heart,kidney, and liver were harvested 2 week after a single injection. TissueGb3 levels were analyzed by mass-spectrometry as described (Durant, B.et al. Sex differences of urinary and kidney globotriaosylceramide andlysoglobotriaosylceramide in Fabry mice. J Lipid Res 52, 1742-1746(2011), incorporated herein by reference in its entirety).

Example 13—Role of Dose of GLA Enzyme for Biodistribution and SubstrateRemoval in Mouse Model of Fabry Disease

To investigate the role dose plays in the biodistribution andtherapeutic effect of our glycoengineered GLA enzymes, we used the Fabrymouse model. Fabrazyme and GLA-bi2,3SA was injected intravenously intoFabry mice in doses of 0.5 mg/kg and 0.2 mg/kg and enzymebiodistribution and activity was analyzed as described below.

Experiment 1—Biodistribution of GLA-bi23SA and Fabrazyme given at 0.5and 0.2 mg/kg dose in Fabry mice: The methods used were as described inExample 3 except that organs were collected after 1 week. Enzymepreparations were injected into female Fabry mice (˜5 months old) at adose of 0.2 or 0.5 mg/kg. Heart, kidney, and liver were dissected at 1week after a single injection. FIG. 23 , reproduced from Tian et al.2019, shows relative distribution of GLA variants into heart, kidney andliver. These data confirm the improved distribution of GLA-Bi23SA toheart and lower uptake in the liver compared to Fabrazyme, but alsodemonstrate dose dependency, suggesting that further studies of optimaldosing for GLA-Bi23SA may be beneficial.

Experiment 2—Substrate removal effect of GLA-bi23SA and Fabrazymeadministered at 0.5 and 0.2 mg/kg doses in Fabry mice: Heart and Kidneywas analyzed for Gb3 substrate reduction as described in Example 4.GLA-Bi23SA or Fabrazyme enzyme preparations (0.5 or 0.2 mg/kg) orvehicle alone (saline) were injected into 6-month old female Fabry micevia tail-vein (n=5 per group). One week after injection, heart andkidney were dissected, and Gb3 levels were measured usingmass-spectrometry. Results of the Gb3 analysis are shown in FIG. 24 ,reproduced from Tian et al. 2019, and confirm the efficient function ofboth GLA-Bi23SA and Fabrazyme after single dose administration. Thesedata clearly demonstrate that glycoforms of GLA without Man6P areefficiently taken up by cells and delivered to the lysosome, where theyefficaciously function to reduce Gb3.

Example 14—Role of 2.6 Sialic Acid Linkage on Glycoengineered GLA Enzymefor Substrate Removal in Mouse Model of Fabry Disease

To investigate the role of 2,6 sialic acid linkage in the efficacy ofGLA enzyme, in vivo, GLA-26SA or vehicle alone (saline) was injectedinto 6-month old female Fabry mice via tail-vein (n=5 per group). At 2weeks after injection, heart, kidney and liver were dissected. Gb3levels were measured in GLA-26SA mouse tissues, and mock-treated Fabrymouse controls (samples from Example 4). For mock controls, the lysatesfrom Example 4 were thawed, sonicated again and then were subjected toprotein assay and glycolipid extraction together with new GLA-2,6SAsamples. The Gb3 levels of the three organs are presented in FIG. 25 ,where data for Fabrazyme and GLA-bi23SA from Example 4 are included forcomparison. GLA-26SA produced lower reduction of Gb3 levels in the heartand kidney compared to Fabrazyme and GLA-bi23SA, while the effect inliver was similar for all variants. This finding correlates with thelower level of GLA-26SA distributed to the heart and especially kidney(FIG. 6 ) and confirms that the specific type of sialic acid linkage iscritically important for improving biodistribution and biologicaleffects.

Example 15—Role of Glycans on GLA Enzyme for PK in Rat Model of FabryDisease

To demonstrate the kinetics of glycoengineered GLA are notspecies-specific, but are applicable in a second animal, a rat model ofFabry disease was used for the following experiment. The model is basedon a Dark Agouti strain lacking GLA activity as described in Miller2018. The GLA-bi23SA variant was tested and GLA produced in wt cells wasused as control. Enzyme preparations (1 mg/kg) was injectedintravenously into 12- to 14-week old male Fabry rats via tail-vein (n=3for GLA-bi23 SA group and n=1 for control). At time points (5 min, 20min, 60 min, 3 h, and 4 h post-injection), small amounts of bloodsamples were collected from the animals via their tail vein. Plasma wasseparated by centrifugation and was used for enzyme assays (see Example1).

The pharmacokinetics of the GLA-bi23SA showed a clearly prolonged bloodclearance profile (FIG. 26 ) and an increased half-life by three fold(7.6 versus 22.1 min for GLA from wt cells) was observed. Thepharmacokinetics of the GLA-bi23SA modified enzyme in the rat model arevery similar to the kinetics seen in mouse model (Example 3, FIG. 5 )demonstrating cross-species applicability of the glycoengineeredvariants species.

Example 16—Evaluating Pharmacokinetics/Stability of Other LysosomalEnzymes

To characterize the effect of optimized glycans on pharmacokinetics weused balb/c mice were used. The plasma activity of endogenous mouseenzyme is negligible (<1%), compared to activities after intravenousinjection, so enzyme kinetics at early time points may be evaluated inthis model. Various optimized glycans were displayed on AGA, GUSB, Lamanand GLA enzymes by performing gene modifications in COH cell linesstably expressing the enzymes. The enzymes produced in wild-type (wt)CHO cells are included for control. The enzymes and glycodesignsevaluated are shown in Table 10:

TABLE 10 Glycodesigns of modified lysosomal enzymes Enzyme variantDesign/Glycostructure Cell engineering GLA-opt No Man6P, High-antennary,KO: Gnptab, Mgat4b/5 high 2,3SA KI: ST3GAL4/MGAT4A/5 GLA wt No AGA-optNo Man6P, High 2,3SA KO: Gnptab KI: ST3GAL4 AGA wt No GUSB-opt No Man6PKO: Gnptab GUSB wt No Laman-opt No Man6P KO: Gnptab Laman wt No

The enzyme variants were injected into 9-10 weeks old Balb/c mice viatail vein at a dose of 0.75 mg/kg body weight (n=2 for each enzymevariant). Blood samples were collected by cheek bleed at 30 and 120 minfor AGA, GUSB and Laman enzymes (15 and 30 min for GLA). The enzymeactivity in plasma was measured using the following substrates: Asp-AMC(L-Aspartic acid β-(7-amido-4-methylcoumarin) for AGA activity,4-Nitrophenyl β-D-glucuronide for GUSB activity, 4-Nitrophenylα-D-mannopyranoside for Laman activity, and 4-Methylumbelliferylalpha-D-Galactopyranoside for GLA activity. All enzyme activities werecorrected for endogenous activity by subtracting enzyme activity ofplasma from untreated mice. The initial plasma activity was based ontotal enzyme activity infused, which we assumed would initially bepresent in the plasma compartment. Plasma activity at the two timepoints as percentage of initial plasma activity is presented in FIG. 27. For all four enzymes, and all sampling times, the glyco-optimizedvariants from engineered cells showed higher activity in plasma comparedto enzyme produced in unmodified wild-type CHO cells. FIGS. 27B, 27C,and 27D show that the glycoengineering involving removal of Man6Pprolongs the circulation of the three enzymes AGA, GUSB and Laman,respectively. This demonstrates that the optimized glycodesigns arebroadly applicable to lysosomal enzymes. In addition, the optimized GLA(GLA-opt in FIG. 27A) shows that a glycodesign without Man6P and withhigher antennarity and high 2,3SialicAcid prolongs the circulatoryhalf-life of GLA enzyme.

Example 17—Ontimizing GLA Variants Stabilized by Protein Mutagenesis

The glyco-optimization introduced into GLA-bi23SA may be combined withenzyme stabilizing technologies involving amino acid mutations of theGLA sequence to obtain additive or synergistic effects. GLA mutants(mutGLA) with improved thermal or physical stability or improved celluptake have been described in WO 2016/105889, incorporated herein byreference in its entirety.

The mutGLA is glycoengineered using the glycodesigns developed for GLA.

The following engineering is performed in CHO cells expressing mutGLA:

-   -   1) knock-out of Gnptab    -   2) knock-out of Gnptab/St6gal1    -   3) knock-out of Gnptab/St6gal1 and knock-in of ST3GAL4    -   4) knock-out of Gnptg/Gnptab/St6gal1 and knock-in of ST3GAL4    -   5) knock-out of Gnptg/Gnptab/St6 gal/Mgat4B/Mgat5 and knock-in        of ST3GAL4

Cell pools or clonal cell lines are isolated, and glyco-optimized mutGLAvariants are produced and characterized, as described in Examples 1 and2.

Pharmacokinetics of mutGLA-bi23SA and other glycooptimized mutGLAvariants are established by administrating 0.1/0.2/0.5 or 1.0 mg/kg toFabry mice or rats. Pharmacokinetics are established as described inExample 3.

Tissue distribution of the mutGLA-bi23SA is established 24 h or 48 hafter injection when animals are sacrificed and heart, kidney, liver andspleen are dissected. Biodistribution is determined a described inExample 3.

Substrate reduction effect of glycoengineered mutGLA variants isestablished by injecting GLA or glycooptimized mutGLA variants producedin glycoengineered cell lines (1-5 listed above) to Fabry mice and rats.At 1 or 2 weeks after injection the animals are sacrificed, and organsare collected and analyzed for Gb3 levels. The procedures are asdescribed in Example 4.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, PCT patent application, PCT patent applicationpublications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification or listed inany Application Data Sheet are incorporated herein by reference in theirentirety. From the foregoing it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention.

LIST OF REFERENCES

-   Benjamin E R, Khanna R, Schilling A, Flanagan J J, Pellegrino L J,    Brignol N, Lun Y, Guillen D, Ranes B E, Frascella M, Soska R, Feng    J, Dungan L, Young B, Lockhart D J and Val enzano K J (2012)    Co-administration With the Pharmacological Chaperone A71001    Increases Recombinant Human α-Galactosidase A Tissue Uptake and    Improves Substrate Reduction in Fabry Mice. Mol Ther 20(4):717-726.-   Damme, M. et al. (2015) Chronic enzyme replacement therapy    ameliorates neuropathology in alpha-mannosidosis mice. Ann Clin    Transl Neurol 2, 987-1001-   Desnick R J, Schuchman E H. (2012) Enzyme replacement therapy for    lysosomal diseases: lessons from 20 years of experience and    remaining challenges. Annu. Rev. Genomics Hum. Genet. 13, 307-33510-   Grubb J H, Vogler C, Levy B, Galvin N, Tan Y, Sly W S. (2008)    Chemically modified beta-glucuronidase crosses blood-brain barrier    and clears neuronal storage in murine mucopolysaccharidosis VII.    Proc.Natl.Acad. Sci USA105:2616-2621-   Grubb, J. H., Vogler, C. & Sly, W. S. New strategies for enzyme    replacement therapy for lysosomal storage diseases. Rejuvenation Res    13, 229-236 (2010)-   Kishnani P. S. (2015) Challenges of Enzyme Replacement Therapy: Poor    Tissue Distribution in Lysosomal Diseases Using Pompe Disease as a    Model. In: Rosenberg A., Demeule B. (eds) Biobetters. AAPS Advances    in the Pharmaceutical Sciences Series, vol 19. Springer, New York,    N.Y.-   Kim J P, Olson L J, Dahms (2009) Carbohydrate Recognition by the    Mannose 6-phosphate Receptors. Curr Opin Struct Biol 19(5):534-42    (2009)-   Lee K, Jin X, Zhang K, Copertino L, Andrews L, Baker-Malcolm J,    Geagan L, Qiu H, Seiger K, Barngrover D, McPherson J M,    Edmunds T. (2003) A biochemical and pharmacological comparison of    enzyme replacement therapies for the glycolipid storage disorder    Fabry disease. Glycobiology 13(4): 305-313-   Lonowski, L A, Narimatsu Y, Riaz A, Delay C E, Yang Z, Niola F, Duda    K, Ober E A, Clausen H, Wandall H H, Hansen S H, Bennett E P,    Frodin M. (2017) Genome editing using FACS enrichment of    nuclease-expressing cells and indel detection by amplicon analysis.    Nature Protocols 12, 581-603 (2017).-   Miller J J, Aoki K, Moehring F, Murphy C A, O'Hara C L, Tiemeyer M,    Stucky C L, Dahms N M (2018) Neuropathic pain in a Fabry disease rat    model. JCI Insight; 3(6). pii: 99171-   Narimatsu Y, Joshi H J, Zhang Y, Gomes C, Chen Y H, Lorenzetti F,    Furukawa S, Schjoldager K, Hansen L, Clausen H, Bennett E P, Wandall    H H (2018): A validated gRNA libraryfor CRISPR/Cas9 targeting of the    human glycosyltransferase genome. Glycobiology 28(5):295-305.-   Platt F M (2018) Emptying the stores: lysosomal diseases and    therapeutic strategies Nat Rev Drug Disc 17: 133-150.-   Rozaklis T, Beard H, Hassiotis S, Garcia A R, Tonini M, Luck A, Pan    J, Lamsa J C, Hopwood J J, Hemsley K M (2011) Impact of high-dose,    chemically modified sulfamidase on pathology in a murine model of    MPS IIIA. Experimental Neurology 230:123-130.-   Shen, J. S. et al. (2016) Mannose receptor-mediated delivery of    moss-made alpha-galactosidase A efficiently corrects enzyme    deficiency in Fabry mice. J Inherit Metab Dis 39, 293-303.-   Tian W, Ye Z, Wang S, Schulz M A, Coillie J V, Sun L, Chen Y H,    Narimatsu Y, Hansen L, Kristensen C, Mandel U, Bennett E P,    Jabbarzadeh-Tabrizi S, Schiffmann R, Shen J S, Vakhrushev S, Clausen    H, Yang Z (2019): The glycosylation design spacefor recombinant    lysosomal replacement enzymes produced in CHO cells. Nature    Communications 10 (1785). DOI: 10.1038/s41467-019-09809-3-   Vakhrushev S Y, Dadimov D, Peter-Katalinic J. (2009) Software    platform for high-throughput glycomics. Anal Chem 81, 3252-3260-   Xu S et al (2015) Coformulation of a Novel Human α-Galactosidase A    With the Pharmacological Chaperone AT1001 Leads to Improved    Substrate Reduction in Fabry Mice. Molecular Therapy vol. 23 no. 7,    1169-1181-   Yang Z., Wang S, Halim A, Schulz M A, Frodin M, Rahman S H,    Vester-Christensen M B, Behrens C, Kristensen C, Vakhrushev S Y,    Bennett E P, Wandall H H, and Clausen H. (2015) Engineered CHO cells    for production of diverse, homogeneous glycoproteins. Nature    biotechnology 33, 842-844-   Zhu Y, Li X, Kyazike J, Zhou Q, Thurberg B L, Raben N, Mattaliano R    J and Cheng S H. (2004) Conjugation of Mannose    6-Phosphate-containing Oligosaccharides to Acid—Glucosidase Improves    the Clearance of Glycogen in Pompe Mice. The Journal of Biological    Chemistry 279: 50336-50341-   Essentials of Glycobiology. 2017, 3^(rd) edition. Varki A, Cummings    R D, Esko J D, et al, editors. Cold Spring Harbor (N. Y.): Cold    Spring Harbor Laboratory Press;

PATENT LITERATURE

-   U.S. Pat. No. 7,001,994 Methods for introducing Mannose 6 Phosphatre    and other oligosaccharides onto Glycoproteins. Zhy Y (Genzyme)-   WO2008/109677 Modified Enzyme and Treatment Method, Sly W S, Grubb J    H, Vogler C A (St. Louis University)-   WO 2015/150490 Modified Sulfamidase and Production Thereof, Berghard    C, Nordling E, Svensson S G, Tjernberg A. (SOBI)-   WO2017/194699 A cell-based array platform, Bennet E, Narimatsu Y,    Steentoft C, Yang Z, Mandel U, Clausen H. (U Copenhagen)-   WO2016091268 N-Glycosylation Rahman S H, Behrens C,    Vester-Christensen M B, Clausen H, Zhang Y, Halim A F, Bennett E (U    Copenhagen, Novo Nordisk A/S)-   WO2017008982 Production of n-glycoproteins for enzyme assisted    glycomodification Rahman S H, Behrens C, Vester-Christensen M B,    Clausen H, Zhang Y, Halim A F, Bennett E (U Copenhagen, Novo Nordisk    A/S)-   WO 2016/105889 Human Alpha-Galactosidase variants Agard N J, Miller    M G, Zhang X, Huisman, GW. (Codexis Inc)

The invention claimed is:
 1. A modified recombinant lysosomal enzymewith increased circulation time in plasma as compared to an unmodifiedversion of the same, wherein said enzyme comprises less than 10%mannose-6-phosphate (Man6P) and less than 0.3 mole exposed mannose (Man)per mole of enzyme, and wherein said enzyme comprises more than 4 molesalpha2,3 sialic acid (SA) per mole of enzyme, and wherein the modifiedrecombinant lysosomal enzyme is Alpha-Galactosidase A (GLA) orAspartvlglucoaminidase (AGA).
 2. The modified recombinant lysosomalenzyme of claim 1, comprising more than 4.5 mol alpha2,3 SA per mol ofenzyme.
 3. The modified recombinant lysosomal enzyme of claim 1,comprising more than 4 mol alpha2,3 SA per mol of enzyme and less than 1mol alpha2,6SA.
 4. The modified recombinant lysosomal enzyme of claim 1,wherein the modified recombinant lysosomal enzyme comprises nodetectable M6P, and high 2,3SA.
 5. The modified recombinant lysosomalenzyme of claim 4, wherein the modified recombinant lysosomal enzymecomprises a biantennary N glycan structure, a triantennary N glycanstructure, a tetra antennary N glycan structure, or combinationsthereof.
 6. The modified recombinant lysosomal enzyme of claim 1,wherein said modified recombinant lysosomal enzyme comprises loweredmannose-6- phosphate (M6P) tagging of N-glycans as compared to a similarunmodified recombinant lysosomal enzyme.